This was confirmed by experiments in which following inhibitor Pfizer MyD88 knockdown (controls in Supplementary Figure 3B, available online), apoptosis is shown to be independent of the presence or absence of p65 (Figure 3C). In contrast, treatment of HCT116 p53+/+ cells with the MEK inhibitor U0126 induced apoptosis (data not shown), consistent with earlier reports that the inhibition of the canonical Ras pathway induces cell death (9). To directly address the implication of the Ras/Erk pathway in MyD88 silencing-induced apoptosis, we performed complementation assays in which we bypass the inhibition of the Ras pathway induced by MyD88 silencing by concomitantly expressing a constitutively active form of MEK (controls in Supplementary Figure 3C, available online). Figure 3D shows that this statistically significantly reduces apoptosis (22.

0%��1.7% vs 38.1%��3.9%; P = .009), indicating that this apoptosis is at least partly due to the inactivation of the MAP kinase canonical pathway following loss of MyD88. MyD88 and DNA Repair Previous studies have established a link between the inhibition of Ras pathway and the accumulation of DNA damage (10). A direct role for Erk was reported in the transcription of ERCC1 DNA repair enzyme (11). We therefore analyzed the levels of ERCC1 in HCT116 p53+/+ or p53�C/�C cells and observed in both cell lines a decrease in the expression of this enzyme (Figure 4A) upon MyD88 silencing. Next, we reasoned that the down-regulation of a major DNA repair enzyme such as ERCC1 should lead to the accumulation of DNA damage.

Indeed, we show an increase in H2AX S139 phosphorylation��an indicator of DNA damage (12)��in both HCT116 p53+/+ or p53�C/�C cell lines (Figure 4B) upon MyD88 silencing (controls in Supplementary Figure 4A). To address whether ERCC1 downregulation and accumulation of DNA damage are causally related, we performed complementation assays in which the inhibition of ERCC1 production induced by MyD88 silencing is bypassed by reexpressing ERCC1 in HCT116 p53+/+ cells. As shown in Figure 4, ,CC and andD,D, this is indeed the case, as ectopically expressing ERCC1 in the cells in which MyD88 was silenced (controls in Supplementary Figure 4B) statistically significantly reverses both the accumulation of DNA damage (pH2AX positive cells: 8.3%��0.5% vs 11.7%��1.0%; P = .004) and apoptosis (25.1%��3.3% vs 33.8%��1.2%; P = .

008), Batimastat respectively. A statistically substantial level of apoptosis remains in cells complemented with MEK or ERCC1. This could be due to the fact that in these experiments MyD88 inhibition occurred in virtually all cells, which express a stably expressed lentiviral vector, whereas the complementing genes were introduced using lipophilic transfection, which is relatively inefficient��typically in the order of 30%�C50% in our hands. Figure 4. MyD88 silencing, ERCC1, and DNA damage.