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 intel action of transcription factors with their response elements. NF-kappa B is a member of the rel family of transcription factors that regulate transcription of genes in the proliferative/anti-apoptotic pathways. We psrformed electrophoretic mobility shift assays to study the role of polyamines in NF-kappa B binding to NF-kappa B 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-kappa B to NRE in the absence of polyamines. Addition of 1 mM spermidine or spermine caused a 4- and 6-fold increase in NF-kappa B-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-kappa B to be a major component in NF-kappa B-NRE complex formation in the presence of polyamines. However, the decreased intensity of the band corresponding to NF-kappa B-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-kappa B-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-kappa B-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-kappa B driven secreted alkaline phosphatase (SEAP) reporter, spermine induced NF-kappa B activity by similar to 2-fold as compared to controls. Spermine induced activation of NF-kappa B was also confirmed using an NF-kappa B-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-kappa B to NRE and a possible mechanism for the action of polyamines in breast cancer cell proliferation.