Epigenetic silencing of RASSF (Ras association domain family) genes RASSF1 and RASSF5 (also called NORE1) by CpG hypermethylation is found frequently in many cancers. Although the physiological roles of RASSF1 have been studied in some detail, the exact functions of RASSF5 are not well understood. Here, we show that RASSF5 plays an important role in mediating apoptosis in response to death receptor ligands, TNF-␣ and TNF-related apoptosis-inducing ligand. Depletion of RASSF5 by siRNA significantly reduced TNF-␣-mediated apoptosis, likely through its interaction with proapoptotic kinase MST1, a mammalian homolog of Hippo. Consistent with this, siRNA knockdown of MST1 also resulted in resistance to TNF-␣-induced apoptosis. To further study the role of Rassf5 in vivo, we generated Rassf5-deficient mouse. Inactivation of Rassf5 in mouse embryonic fibroblasts (MEFs) resulted in resistance to TNF-␣-and TNF-related apoptosis-inducing ligandmediated apoptosis. Importantly, Rassf5-null mice were significantly more resistant to TNF-␣-induced apoptosis and failed to activate Mst1. Loss of Rassf5 also resulted in spontaneous immortalization of MEFs at earlier passages than the control MEFs, and Rassf5-null immortalized MEFs, but not the immortalized wild type MEFs, were fully transformed by K-RasG12V. Together, our results demonstrate a direct role for RASSF5 in death receptor ligand-mediated apoptosis and provide further evidence for RASSF5 as a tumor suppressor.A small family of genes termed RASSF (Ras association domain family) has been recently described, the members of which are characterized by the presence of a Ras association (RA) 4 domain and a novel motif named the SARAH (Salvador, Rassf, Hippo) domain at the C terminus (reviewed in Refs. 1-3). Among the members of the RASSF family, RASSF1 and RASSF5 (also known as NORE1, for novel Ras effector 1) share the closest homology, displaying 49% identity (66% similarity) at the protein level, and are frequently inactivated by CpG hypermethylation in human cancer cell lines and primary tumors (4 -6). RASSF1 has been studied extensively (reviewed in Ref. 7) and shown to play important roles in mitosis (8), microtubule and genomic stability (9 -11), apoptosis (12, 13), and cell cycle (14), which are consistent with its function as a tumor suppressor. However, not much is known about the physiological roles of RASSF5.RASSF5 encodes multiple isoforms because of dual promoter usage and alternative splicing (15). The longest isoform, designated RASSF5A (NORE1A), is transcribed from the 5Ј-most promoter region containing CpG island and encodes a 418-amino acid protein containing the cysteine-rich diacylglycerol/ phorbol ester-binding domain (also called protein kinase C conserved region 1, C1), the RA domain, and the C-terminal SARAH domain. Through alternative splicing, another isoform RASSF5B (NORE1A␤), lacking the SARAH domain is produced. The shortest isoform, RASSF5C (NORE1B, also called RAPL), lacking the N-terminal C1 domain, is produced from a downstream CpG-containing ...

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Although it has been suggested that kinesin family member 14 (KIF14) has oncogenic potential in various cancers, including hepatocellular carcinoma (HCC), the molecular mechanism of this potential remains unknown. We aimed to elucidate the role of KIF14 in hepatocarcinogenesis by knocking down KIF14 in HCC cells that overexpressed KIF14. After KIF14 knockdown, changes in tumor cell growth, cell cycle and cytokinesis were examined. We also examined cell cycle regulatory molecules and upstream Skp1/Cul1/F-box (SCF) complex molecules. Knockdown of KIF14 resulted in suppression of cell proliferation and failure of cytokinesis, whereas KIF14 overexpression increased cell proliferation. In KIF14-silenced cells, the levels of cyclins E1, D1 and B1 were profoundly decreased compared with control cells. Of the cyclin-dependent kinase inhibitors, the p27Kip1 protein level specifically increased after KIF14 knockdown. The increase in p27Kip1 was not due to elevation of its mRNA level, but was due to inhibition of the proteasome-dependent degradation pathway. To explore the pathway upstream of this event, we measured the levels of SCF complex molecules, including Skp1, Skp2, Cul1, Roc1 and Cks1. The levels of Skp2 and its cofactor Cks1 decreased in the KIF14 knockdown cells where p27Kip1 accumulated. Overexpression of Skp2 in the KIF14 knockdown cells attenuated the failure of cytokinesis. On the basis of these results, we postulate that KIF14 knockdown downregulates the expression of Skp2 and Cks1, which target p27Kip1 for degradation by the 26S proteasome, leading to accumulation of p27Kip1. The downregulation of Skp2 and Cks1 also resulted in cytokinesis failure, which may inhibit tumor growth. To the best of our knowledge, this is the first report that has identified the molecular target and oncogenic effect of KIF14 in HCC.

Treatment of L1210 cells with prostaglandin A2 (PGA2) or 9‐deoxy‐Δ9,12‐13,14‐dihydro PGD2 (Δ12‐PGJ2) resulted in significant G2/M arrest and subsequent DNA fragmentation at concentrations that are cytotoxic to the cells. On agarose gel electrophoresis, DNA ladder formation was evident 24 h after the addition of Δ12‐PGJ2 and remained apparent through 72 h, whereas G2/M accumulation was observed 6 h after the treatment. When the morphology of cells was examined by electron microscopy, L1210 cells incubated with a cytotoxic dose of PGA2 or Δ12‐PGJ2 for 24 h showed the characteristic morphological features of apoptosis such as chromatin condensation, nuclear fragmentation and formation of apoptotic body. Cycloheximide blocked the DNA fragmentation and morphological changes induced by Δ12‐PGJ2. Our results suggest that these cyclopentenone PGs caused apoptotic cell death of L1210 cells which is preceded by G2/M accumulation and requires de novo protein synthesis.

p300/CBP-associated factor (PCAF), a histone acetyltransferase, is involved in many cellular processes such as differentiation, proliferation, apoptosis, and reaction to cell damage by modulating the activities of several genes and proteins through the acetylation of either the histones or transcription factors. Here, we examined a pathogenic role of PCAF and its potential as a novel therapeutic target in the progression of renal tubulointerstitial fibrosis induced by non-diabetic unilateral ureteral obstruction (UUO) in male C57BL/6 mice. Administration of garcinol, a PCAF inhibitor, reversed a UUO-induced increase in the renal expression of total PCAF and histone 3 lysine 9 acetylation and reduced positive areas of trichrome and α-smooth muscle actin and collagen content. Treatment with garcinol also decreased mRNA levels of transforming growth factor-β, matrix metalloproteinase (MMP)-2, MMP-9, and fibronectin. Furthermore, garcinol suppressed nuclear factor-κB (NF-κB) and pro-inflammatory cytokines such as tumor necrosis factor-α and IL-6, whereas it preserved the nuclear expression of nuclear factor erythroid-derived 2-like factor 2 (Nrf2) and levels of Nrf2-dependent antioxidants including heme oxygense-1, catalase, superoxide dismutase 1, and NAD(P)H:quinone oxidoreductase 1. These results suggest that the inhibition of inordinately enhanced PCAF could mitigate renal fibrosis by redressing aberrant balance between inflammatory signaling and antioxidant response through the modulation of NF-κB and Nrf2.