Jaejoon Won

Hexachlorophene inhibits Wnt/beta-catenin pathway by promoting Siah-mediated beta-catenin degradation.

Aberrant activation of Wnt/β-catenin signaling and subsequent up-regulation of β-catenin response transcription (CRT) is a critical event in the development of human colon cancer. Thus, Wnt/β-catenin signaling is an attractive target for the development of anticancer therapeutics. In this study, we identified hexachlorophene as an inhibitor of Wnt/β-catenin signaling from cell-based small-molecule screening. Hexachlorophene antagonized CRT that was stimulated by Wnt3a-conditioned medium by promoting the degradation of β-catenin. This degradation pathway is Siah-1 and adenomatous polyposis colidependent, but glycogen synthase kinase-3β and F-box β-transducin repeat-containing protein-independent. In addition, hexachlorophene represses the expression of cyclin D1, which is a known β-catenin target gene, and inhibits the growth of colon cancer cells. Our findings suggest that hexachlorophene attenuates Wnt/β-catenin signaling through the Siah-1-mediated β-catenin degradation.

Protein-kinase-C-mediated β-catenin phosphorylation negatively regulates the Wnt/β-catenin pathway

Normally, the Wnt/β-catenin pathway controls developmental processes and homeostasis, but abnormal activation of this pathway is a frequent event during the development of cancer. The key mechanism in regulation of the Wnt/β-catenin pathway is the amino-terminal phosphorylation of β-catenin, marking it for proteasomal degradation. Here we present small-molecule-based identification of protein kinase C (PKC)-mediated β-catenin phosphorylation as a novel mechanism regulating the Wnt/β-catenin pathway. We used a cell-based chemical screen to identify A23187, which inhibits the Wnt/β-catenin pathway. PKC was activated by A23187 treatment and subsequently phosphorylated N-terminal serine (Ser) residues of β-catenin, which promoted β-catenin degradation. Moreover, the depletion of PKCα inhibited the phosphorylation and degradation of β-catenin. Therefore, our findings suggest that the PKC pathway negatively regulates the β-catenin level outside of the Wnt/β-catenin pathway.

Opposing regulatory roles of E2F in human telomerase reverse transcriptase (hTERT) gene expression in human tumor and normal somatic cells

Telomerase activity is closely correlated with cellular proliferative activity in human tissues. Human cells with high proliferative potential, such as tumor cells or stem cells, exhibit telomerase activity, whereas most normal human somatic cells do not. Telomerase activity is tightly regulated by the expression of its catalytic subunit human telomerase reverse transcriptase (hTERT). Through an expression cloning approach, we identified E2F‐1 as a repressor of the hTERT gene in human tumor cells. Ectopic expression of E2F‐1 repressed hTERT promoter activity by inhibiting Sp1 activation of the hTERT promoter. In contrast to the repressor function of E2F‐1 in human tumor cells, we demonstrated that E2F‐1 is an activator of the hTERT gene in normal human somatic cells. Ectopically expressed E2F‐1 activated the hTERT promoter through a noncanonical DNA binding site. E2F‐1, E2F‐2, and E2F‐3 (but not E2F‐4 and E2F‐5) repressed hTERT promoter activity in human tumor cells, whereas they activated it in normal somatic cells. These contrasting effects of E2F transcription factors on the hTERT promoter could underlie the paradoxical biological activities of E2F, which can both promote and inhibit cellular proliferation and tumorigenesis.

Diclofenac attenuates Wnt/β-catenin signaling in colon cancer cells by activation of NF-κB

The dysregulation of Wnt/β‐catenin signaling and subsequent upregulation of β‐catenin response transcription (CRT) occur frequently in colon cancer cells. Non‐steroidal anti‐inflammatory drugs (NSAIDs) can repress CRT in colorectal cancer, but little is known about the mechanism of action. We show that the NSAID diclofenac inhibits Wnt/β‐catenin signaling without altering the level of β‐catenin protein and reduces the expression of β‐catenin/TCF‐dependent genes. Diclofenac induced the degradation of IκBα, which increased free nuclear factor κB (NF‐κB) in cells. Also, the ectopic expression of p65, which is a component of NF‐κB, suppressed CRT. Our findings suggest that diclofenac inhibits Wnt/β‐catenin signaling via the activation of NF‐κB in colon cancer cells.

Retraction: Small molecule–based reversible reprogramming of cellular lifespan

In this paper, we described the application of magnetism-based interaction capture (MAGIC) to find a small-molecule protein target. MAGIC identified the protein ATM as the target of CGK733, a small molecule reportedly isolated by high-throughput screening.

Histone Modifications and Transcription Factor Binding on Chromatin: ChIP–PCR Assays

Chromatin, the eukaryotic template of genetic information, is subject to a diverse array of posttranslational modifications that largely impinge on the N-termini of histones, such as acetylation, methylation, phosphorylation, and ubiquitination. Distinct histone modifications generate synergistic or antagonistic interaction affinities for novhistone proteins, which in turn dictate dynamic transitions between transcriptionally active or silent states of chromatin. Besides transcription, numerous biological processes, including DNA replication, DNA repair, and recombination, are regulated by chromatin-associated factors. The chromatin immunoprecipitation (ChIP) technique provides us with an exquisite tool to investigate the interplay between the structural or regulatory proteins and DNA and its role in regulating diverse cellular processes in vivo by formaldehyde crosslinking of proteins to proteins and proteins to DNA, followed by immunoprecipitation of the fixed material and detection of the associated DNA. Here we illustrate the overall experimental procedure by taking ChIP analysis of the human telomerase reverse transcriptase promoter as an example.