Activation of nuclear factor kappaB 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-kappaB 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-kappaB binding to NF-kappaB 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-kappaB to NRE in the absence of polyamines. Addition of 1 mM spermidine or spermine caused a 4- and 6-fold increase in NF-kappaB-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-kappaB to be a major component in NF-kappaB-NRE complex formation in the presence of polyamines. However, the decreased intensity of the band corresponding to NF-kappaB-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-kappaB-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-kappaB-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-kappaB driven secreted alkaline phosphatase (SEAP) reporter, spermine induced NF-kappaB activity by approximately 2-fold as compared to controls. Spermine induced activation of NF-kappaB was also confirmed using an NF-kappaB-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-kappaB to NRE and a possible mechanism for the action of polyamines in breast cancer cell proliferation.
Shah
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Department of Medicine, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, New Brunswick 08903, USA.
Thomas
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T
Shirahata
A
A
Sigal
L H
LH
Thomas
T J
TJ
D001943:Breast Neoplasms / Q000378:metabolism*; D002942:Circular Dichroism; D005260:Female; D006146:Guanidines / Q000494:pharmacology; D006801:Humans; D016328:NF-kappa B / Q000378:metabolism*; D011073:Polyamines / Q000494:pharmacology*; D011487:Protein Conformation; D011700:Putrescine / Q000494:pharmacology; D013095:Spermidine / Q000494:pharmacology; D013096:Spermine / Q000494:pharmacology; D013329:Structure-Activity Relationship; D014407:Tumor Cells, Cultured
D006146:Guanidines; D016328:NF-kappa B; D011073:Polyamines; D013096:Spermine; C004479:pimagedine; D013095:Spermidine; D011700:Putrescine