Hiroki Kamino

Possible Role of Semaphorin 3F, a Candidate Tumor Suppressor Gene at 3p21.3, in p53-Regulated Tumor Angiogenesis Suppression

Although the regulation of tumor angiogenesis is believed to be one of the core functions of p53, the mechanism still remains to be elucidated. Here, we report that semaphorin 3F (SEMA3F), an axon guidance molecule, is involved in p53-regulated antiangiogenesis. The expression level of SEMA3F mRNA was increased by both exogenous and endogenous p53. Chromatin immunoprecipitation assay indicated that a potent p53-binding sequence in intron 1 of SEMA3F interacts with p53 and that it has a p53-responsive transcriptional activity. Overexpression of SEMA3F inhibited in vitro cell growth of the lung cancer cell line H1299. In nude mice assay, the size of the H1299 tumors expressing SEMA3F was much smaller, and they showed lesser number of blood vessels as compared with the control tumors. Moreover, tumors derived from the p53-knockdown colorectal cancer cell line LS174T displayed a remarkable enhancement of tumor vessel formation as compared with control tumors containing normal levels of p53. The expression levels of SEMA3F and neuropilin-2 (NRP2), the functional receptor for SEMA3F, in p53-knockdown LS174T tumors were lower than those in the control tumors. Adenovirus-mediated SEMA3F gene transfer induced the remarkable in vitro growth suppression of the stable transformant of H1299 cells, which express high levels of NRP2. These results suggest that p53 negatively regulates tumor vessel formation and cell growth via the SEMA3F-NRP2 pathway.

The potential role of DFNA5, a hearing impairment gene, in p53-mediated cellular response to DNA damage

AbstractThe tumor suppressor p53 plays a crucial role in the cellular response to DNA damage by transcriptional activation of numerous downstream genes. Although a considerable number of p53 target genes have been reported, the precise mechanism of p53-regulated tumor suppression still remains to be elucidated. Here, we report a novel role of the DFNA5 gene in p53-mediated etoposide-induced cell death. The DFNA5 gene has been previously reported to be responsible for autosomal-dominant, nonsyndromic hearing impairment. The expression of the DFNA5 gene was strongly induced by exogenous and endogenous p53. The chromatin immunoprecipitation assay indicated that a potential p53-binding sequence is located in intron 1 of the DFNA5 gene. Furthermore, the reporter gene assay revealed that the sequence displays p53-dependent transcriptional activity. The ectopic expression of DFNA5 enhanced etoposide-induced cell death in the presence of p53; however, it was inhibited in the absence of p53. Finally, the expression of DFNA5 mRNA was remarkably induced by gamma-ray irradiation in the colon of p53(+/+) mice but not in that of p53(−/−) mice. These results suggest that DFNA5 plays a role in the p53-regulated cellular response to genotoxic stress probably by cooperating with p53.

BNIP3 and NIX Mediate Mieap-Induced Accumulation of Lysosomal Proteins within Mitochondria

Mieap, a p53-inducible protein, controls mitochondrial quality by repairing unhealthy mitochondria. During repair, Mieap induces the accumulation of intramitochondrial lysosomal proteins (designated MALM for Mieap-induced accumulation of lysosome-like organelles within mitochondria) by interacting with NIX, leading to the elimination of oxidized mitochondrial proteins. Here, we report that an additional mitochondrial outer membrane protein, BNIP3, is also involved in MALM. BNIP3 interacts with Mieap in a reactive oxygen species (ROS)-dependent manner via the BH3 domain of BNIP3 and the coiled-coil domains of Mieap. The knockdown of endogenous BNIP3 expression severely inhibited MALM. Although the overexpression of either BNIP3 or NIX did not cause a remarkable change in the mitochondrial membrane potential (MMP), the co-expression of all three exogenous proteins, Mieap, BNIP3 and NIX, caused a dramatic reduction in MMP, implying that the physical interaction of Mieap, BNIP3 and NIX at the mitochondrial outer membrane may regulate the opening of a pore in the mitochondrial double membrane. This effect was not related to cell death. These results suggest that two mitochondrial outer membrane proteins, BNIP3 and NIX, mediate MALM in order to maintain mitochondrial integrity. The physical interaction of Mieap, BNIP3 and NIX at the mitochondrial outer membrane may play a critical role in the translocation of lysosomal proteins from the cytoplasm to the mitochondrial matrix.

Identification of p53-46F as a super p53 with an enhanced ability to induce p53-dependent apoptosis.

More than half of human cancers contain mutations in the tumor suppressor protein p53, most of which accumulate in the DNA binding domain of the protein. Here we report the identification of a mutant p53, designated p53–46F, in which Ser‐46 is replaced with phenylalanine. In vitro, adenovirus‐mediated transduction of the p53–46F gene induced apoptosis more efficiently than wild‐type p53 in a number of cancer cell lines, whereas Ser‐15 phosphorylation of p53–46F was enhanced in all cancer cell lines examined. Moreover, the expression level of the cell cycle inhibitor p21/WAF1 was decreased in cell lines infected with adenovirus p53–46F (Ad‐p53–46F). p53–46F caused a more enhanced level of transcriptional activation of several p53‐target genes, including Noxa, p53AIP1 and p53RFP, compared with wild‐type p53. In vivo, adenovirus‐mediated gene transfer of p53–46F enhanced apoptosis, thus suppressing tumor growth of a lung cancer cell line more effectively than wild‐type p53 or p53–121F, another p53 mutant. Collectively, our data suggest that p53–46F is an active version of p53 that demonstrates enhanced induction of p53‐dependent apoptosis. This is probably mediated by upregulated transactivation of genes downstream of p53, increased Ser‐15 phosphorylation and a decrease in p21/WAF1 levels. We propose p53–46F as an alternative candidate to wild‐type p53 for use in developing new therapeutic strategies for the treatment of cancer. (Cancer Sci 2006; 97: 633–641)

The Nuclear Receptors Constitutive Active/Androstane Receptor and Pregnane X Receptor Activate the Cyp2c55 Gene in Mouse Liver

Mouse CYP2C55 has been characterized as an enzyme that catalyzes synthesis of 19-hydroxyeicosatetraenoic acid (19-HETE), an arachidonic acid metabolite known to have important physiological functions such as regulation of renal vascular tone and ion transport. We have now found that CYP2C55 is induced by phenobarbital (PB) and pregnenolone 16α-carbonitrile (PCN) in both mouse kidney and liver. The nuclear xenobiotic receptors constitutive active/androstane receptor (CAR) and pregnane X receptor (PXR) regulate these drug inductions: CYP2C55 mRNA was increased 25-fold in PB-treated Car(+/+) but not in Car(−/−) mice and was induced in Pxr(+/+) but not Pxr(−/−) mice after PCN treatment. Cell-based promoter analysis and gel shift assays identified the DNA sequence −1679TGAACCCAGTTGAACT−1664 as a DR4 motif that regulates CAR- and PXR-mediated transcription of the Cyp2c55 gene. Chronic PB treatment increased hepatic microsomal CYP2C55 protein and serum 19-HETE levels. These findings indicate that CAR and PXR may play a role in regulation of drug-induced synthesis of 19-HETE in the mouse.

Identification of 14-3-3γ as a Mieap-interacting protein and its role in mitochondrial quality control

Mieap, a p53-inducible protein, controls mitochondrial integrity by inducing the accumulation of lysosomal proteins within mitochondria. This phenomenon is designated MALM, for Mieap-induced accumulation of lysosome-like organelles within mitochondria. To identify this novel Mieap-interacting protein(s), we performed a two-dimensional image-converted analysis of liquid chromatography and mass spectrometry (2DICAL) on the proteins immunoprecipitated by an anti-Mieap antibody. We indentified 14-3-3γ as one of the proteins that was included in the Mieap-binding protein complex when MALM was induced. The interaction between Mieap and 14-3-3γ was confirmed on the exogenous and endogenous proteins. Interestingly, 14-3-3γ was localized within mitochondria when MALM occurred. A 14-3-3γ deficiency did not affect the accumulation of Mieap and lysosomal proteins within mitochondria, but dramatically inhibited the elimination of oxidized mitochondrial proteins. These results suggest that 14-3-3γ plays a critical role in eliminating oxidized mitochondrial proteins during the MALM process by interacting with Mieap within mitochondria.

Nuclear receptor CAR-regulated expression of the FAM84A gene during the development of mouse liver tumors

The nuclear xenobiotic receptor CAR is a phenobarbital (PB)-activated transcription factor. Using a mouse model of two-step liver tumorigenesis, in which tumor growth was initiated by diethyl nitrosamine (DEN) and promoted by chronic treatment with PB, we previously demonstrated that tumors developed only in the presence of CAR. Here, we have identified the FAM84A (family with sequence similarity 84, member A) gene as a CAR-regulated gene that is over-expressed during development of phenobarbital-promoted mouse liver tumors. FAM84A mRNA was induced in the liver of DEN/PB-treated mice prior to the development of liver tumors and this induction continued in the non-tumor as well as tumor tissues of a tumor-bearing liver. Western blotting demonstated that FAM84A protein expression increased in mouse liver after PB treatment; however, the FAM84A protein in liver and liver tumors was not phosphorylated at the serine 38 residue, which has been reported to correlate with morphological changes in cells. Immunohistochemistry analysis revealed the cytoplasmic localization of FAM84A protein and its expression during tumor development in normal tissues (especially in hepatocytes around the central vein), eosinophilic foci, adenomas and carcinomas. HepG2 cell-based reporter assays indicated that CAR activated the FAM84A promoter. Exogenous over-expression of FAM84A in HepG2 cells resulted in increased cell migration. The physiological function of FAM84A remains unknown, but our results suggest that FAM84A is up-regulated by CAR during the development of liver tumors, and may play an important role in the progression of liver cancer by increasing cell migration.

Mieap-regulated mitochondrial quality control is frequently inactivated in human colorectal cancer

Mieap, a p53-inducible protein, controls mitochondrial quality by repairing or eliminating unhealthy mitochondria. BNIP3 and NIX are critical mediators for the Mieap-regulated mitochondrial quality control. Mieap suppresses murine intestinal tumor via its mitochondrial quality control function. To explore the role of the Mieap-regulated mitochondria quality control function in colorectal cancer patients, we examined the statuses of p53, Mieap, BNIP3 and NIX in 57 primary colorectal cancer tissues. Promoter methylation of the Mieap and BNIP3 genes was found in 9% and 47% of colorectal cancer cases, respectively, whereas p53 mutation was found in more than 50% of colorectal cancer tissues lacking methylation of the Mieap and BNIP3 promoters, implying that the p53/Mieap/BNIP3-regulated mitochondria quality control pathway is inactivated in more than 70% of colorectal cancer patients. In LS174T colorectal cancer cells, hypoxia activated the Mieap-regulated mitochondria quality control function. Knockdown of p53, Mieap or BNIP3 in LS174T cells severely impaired the hypoxia-activated function, leading to the accumulation of unhealthy mitochondria and increase of mitochondrial reactive oxygen species generation. The mitochondrial reactive oxygen species generated by unhealthy mitochondria in the p53/Mieap/BNIP3-deficient cells remarkably enhanced cancer cell migration and invasion under hypoxic condition. These results suggest that the Mieap-regulated mitochondria quality control has a critical role in colorectal cancer suppression in the in vivo hypoxic tumor microenvironment.

The nuclear receptor constitutive active/androstane receptor arrests DNA-damaged human hepatocellular carcinoma Huh7 cells at the G2/M phase.

Here, we have demonstrated that xenobiotic activation of the nuclear receptor (CAR, NR1I3) can result in arresting DNA‐damaged human hepatocellular carcinoma Huh7 cells at the G2/M phase. Huh7 cells over‐expressing CAR were either treated with dimethyl sulfoxide, the CAR activator TCPOBOP (1,4‐bis[2‐(3,5‐dichloropyridyloxy)]benzene; androstenol, 16,(5α)‐androsten‐3α‐OL), or repressor androstenol; these treatments were then followed by adriamycin treatment to damage DNA. FACS analysis revealed that CAR‐activation by TCPOBOP increased the rate of arrested Huh7 cells at the G2/M phase (4N DNA content) after DNA damage by adriamycin. This increase correlated with the increase of cell viability in TCPOBOP‐treated Huh7 cells, as determined by MTT assays. Real‐time polymerase chain reaction analysis determined that, as regulated by CAR, the growth arrest and DNA damage‐inducible γ (GADD45γ) and Cyclin G2 genes increased and decreased, respectively, as TCPOBOP increased the number of Huh7 cells arrested at the G2/M phase. Thus, the results suggest that CAR regulates cell cycle, increasing G2/M arrest, and delaying the death of DNA‐damaged cells. Mol. Carcinog.

B-cell linker protein prevents aneuploidy by inhibiting cytokinesis

Aneuploidy is a hallmark of human cancers. Although the maintenance of genomic integrity by p53 is important in preventing aneuploidy, its mechanism remains to be elucidated. Here we report evidence that B‐cell linker protein (BLNK) mediates the inhibition of cytokinesis, which generates tetraploidy but prevents aneuploidy. We identified BLNK as a transcriptional target of p53. Surprisingly, ectopic expression of exogenous BLNK inhibited cytokinesis, resulting in the formation of tetraploid cells. Indeed, BLNK was involved in the generation of spontaneously arising binucleate tetraploid cells. Interestingly, cytokinesis after DNA damage was inhibited in p21−/– and p53+/+ cells, but not in p53−/– cells. BLNK knockdown in p53+/+ and p21−/– cells enhanced cytokinesis after DNA damage, leading to the generation of aneuploid cells. In addition, a BLNK‐downregulated human pre‐B leukemia cell line showed increased cytokinesis and aneuploidy after DNA damage compared with two other pre‐B leukemia cell lines expressing higher levels of BLNK. These results suggest that BLNK acts as a mediator of p53 in the inhibition of cytokinesis, which prevents aneuploidy. We propose that the inhibition of cytokinesis is crucial for the maintenance of genomic integrity. (Cancer Sci 2008; 99: 2444–2454)