Down-regulation of NF-κB activity and NF-κB p65 subunit expression by ras and polyoma middle T oncogenes in human colonic Caco-2 cells

Abstract

The products of ras and src proto-oncogenes are frequently activated in a constitutive state in human colorectal cancer. In this study we attempted to establish whether the tumorigenic progression induced by oncogenic activation of p21 ras or pp60 c-src in human colonic cells is associated with alterations of the activity and expression of nuclear factor kB (NF-kB), a transcription factor suspected to participate in the development of cancer. To this end, we used Caco-2 cells made highly tumorigenic by transfection with an activated Val-12 human Ha-ras gene or with the polyoma middle T (PyMT) oncogene, a constitutive activator of pp60 c-src tyrosine kinase activity. Compared with control vectortransfected Caco-2 cells, both oncogene-transfected cell lines exhibited: (i) decreased constitutive NF-kB DNAbinding activity and NF-kB-mediated reporter gene expression, without alteration of their response to TNF-a for activation of these parameters; (ii) reduced NF-kB cytosolic stores along with a decreased p65 expression due, at least in part, to destabilization of p65 mRNA; (iii) a decrease in adhesion to extracellular matrix component-coated substrata which was partially corrected when stimulating NF-kB transcriptional activity with TNF-a. These results indicate that the tumorigenic progression induced by oncogenic p21 ras or PyMT/pp60 c-src in human colonic Caco-2 cells is associated with a down-regulation of p65 expression and NF-kB activity which could be responsible for the reduced adhesive properties of these cells after oncogene transfection.

Downregulation of the colon tumour-suppressor homeobox gene Cdx-2 by oncogenic ras

“…It is therefore possible that, beside the PKC-mediated e ect, ras additionally alters Cdx-2 gene transcription through PKC-independent pathway(s). In this case, we can exclude the P13K (Phosphatidyl-Inositol-3 kinase) and NF-kB pathways, because the treatment of Caco-2T/MT/MT cells by LY294002 to inhibit P13K activity or by TNFa to restore the NF-kB activity altered by Ha-ras (Cadoret et al, 1997), are ine ective on the expression of Cdx-2 downregulated by oncogenic ras; we also rule out the JNK/SAPK pathway (Jun Nterminal kinase/Stress-Activated-Protein kinase) because JNK activity is unchanged in ras-activated Caco-2T/MT cells compared to control cells (unpublished results).…”

“…EMSA was performed as previously described (Cadoret et al, 1997), with the labelled double-stranded Cdx2-AP1 oligonucleotide (d CGA TTGGTGTCTGTGTCAT-TAC TA ATAGA / dAGACTCTAT TAGTA ATGACACA-GACACCA). Competition with DNA used a 30-fold excess of unlabelled oligonucleotide; the DNA competitors were Cdx2-AP1, Std-AP1 (dCGCTTGATGAGTCAGCCG-GAA/dTTCCG-GCTGACTCATCAAGCG) containing a consensus AP-1 binding site, or OCT-1 (dTGTCGAATG-CAAATCACTAGAA/dTTCTAGTGATTTGCATTCGA-CA) containing an OCT-1 site.…”

Novel Regulatory Action of Ribosomal Inactivation on Epithelial Nod2-Linked Proinflammatory Signals in Two Convergent ATF3-Associated Pathways

“…So far, only a few studies have reported on the regulation of p65 expression at the posttranscriptional level (43,44). Although the ubiquitously encoded housekeeping p65 gene does not contain NF-kB binding motifs in its promoter region (45), the p65 transcript has three AU-rich elements in the 39 untranslated region, which would make the gene susceptible to regulation by RNAstabilizing HuR protein.…”

Insulin Antiapoptotic Signaling Involves Insulin Activation of the Nuclear Factor κB-dependent Survival Genes Encoding Tumor Necrosis Factor Receptor-associated Factor 2 and Manganese-superoxide Dismutase

“…Membranes were hybridized (3 h at 65°C) in Rapid-Hyb buffer (Amersham Pharmacia Biotech) to a random-primed labeled cDNA probe specific for TRAF2, c-IAP1, c-IAP2, or Mn-SOD and subsequently to a labeled human elongation factor 1 probe as a control for RNA loading. Membranes were then washed to high stringency as described (24). Signals were quantified and normalized by differential densitometric scanning of the specific bands versus the human elongation factor 1 band.…”

“…Total RNA (20 g) or poly(A) ϩ RNA (2.5 g) was electrophoresed and transferred to Hybond-N ϩ nylon membranes (Amersham Pharmacia Biotech) as described (24). Membranes were hybridized (3 h at 65°C) in Rapid-Hyb buffer (Amersham Pharmacia Biotech) to a random-primed labeled cDNA probe specific for TRAF2, c-IAP1, c-IAP2, or Mn-SOD and subsequently to a labeled human elongation factor 1 probe as a control for RNA loading.…”