Abstract: Although it has been shown that the gastric tumor suppressor RUNX3 has a growth inhibitory activity, the precise molecular mechanisms behind RUNX3-mediated tumor suppression remained unclear. In this study, we found that RUNX3 is closely involved in DNA damage-dependent phosphorylation of tumor suppressor p53 at Ser-15 and acts as a co-activator for p53. The small interference RNA-mediated knockdown of RUNX3 inhibited adriamycin (ADR)-dependent apoptosis in p53-proficient cells but not in p53-deficient cells in association with a significant reduction of p53-target gene expression as well as phosphorylation of p53 at Ser-15. In response to ADR, RUNX3 was induced to accumulate in the cell nucleus and co-localized with p53. Immunoprecipitation experiments demonstrated that RUNX3 forms a complex with p53 in cells. In vitro pulldown assays revealed that the COOH-terminal portion of p53 is required for the interaction with RUNX3. Forced expression of RUNX3 enhanced p53-mediated transcriptional activation. Additionally, RUNX3 had an ability to induce the phosphorylation of p53 at Ser-15, thereby promoting p53-dependent apoptosis. Intriguingly, RUNX3 interacted with phosphorylated forms of ataxia telangiectasia-mutated in response to ADR; however, it did not affect the extent of DNA damage. From the clinical point of view, coordinated p53 mutation and decreased expression of RUNX3 in 105 human lung adenocarcinomas were significantly associated with the poor outcome of patients (p = 0.0203). Thus, our present results strongly suggest that RUNX3 acts as a novel co-activator for p53 through regulating its DNA damage-induced phosphorylation at Ser-15 and also provide a clue to understanding the molecular mechanisms underlying RUNX3-mediated tumor suppression.
Abstract: NFBD1/MDC1 is a large nuclear protein involved in the early cellular response to DNA damage. Upon DNA damage, NFBD1 has an ability to facilitate the efficient DNA repair. In the present study, we have found that, in addition to DNA damage response, NFBD1 plays a critical role in the regulation of G2/M transition. Expression study using synchronized HeLa cells demonstrated that, like the mitotic kinase Plk1, NFBD1 expression level is maximal in G2/M-phase of the cell cycle. siRNA-mediated knockdown of NFBD1 resulted in G2/M arrest as well as simultaneous apoptosis in association with a significant increase in the amounts of gammaH2AX and pro-apoptotic p73. Since a remarkable down-regulation of mitotic phospho-histone H3 was detectable in NFBD1-knocked down cells, it is likely that knocking down of NFBD1 inhibits G2/M transition. Taken together, our present findings suggest that NFBD1 has a pivotal role in the regulation of proper mitotic entry.
Abstract: MYCN oncogene is one of the most important regulators affecting the prognosis of neuroblastoma and is frequently amplified in the high-risk subsets. Despite its clinical significance, it remains unclear how the MYCN expression is regulated in human neuroblastomas. Here, we found the presence of a positive auto-regulatory mechanism of MYCN. Enforced expression of MYCN induced endogenous MYCN mRNA expression in SK-N-AS neuroblastoma cells with a single copy of MYCN gene. Luciferase reporter assay revealed that MYCN protein activates its own promoter activity in a dose-dependent manner and the downstream region relative to the transcription start sites is responsible for the activation. Furthermore, ChIP analysis showed that MYCN is directly recruited onto the intron 1 region of MYCN gene which contains two putative E-box sites. Intriguingly, in response to all-trans-retinoic acid (ATRA), MYCN was down-regulated in MYCN-amplified SK-N-BE neuroblastoma cells, and the recruitment of MYCN protein onto its own intron 1 region was reduced in association with an induction of neuronal differentiation. Collectively, our present results suggest that MYCN contributes to its own expression by forming a positive auto-regulatory loop in neuroblastoma cells.
Abstract: We previously found that Plk1 inhibited the p53/p73 activity through its direct phosphorylation. In this study, we investigated the functional role of Plk1 in modulating the p53 family member TAp63, resulting in the control of apoptotic cell death in liver tumor cells. Immunoprecipitation and in vitro pull-down assay showed that p63 binds to the kinase domain of Plk1 through its DNA-binding region. in vitro kinase assay indicated that p63 is phosphorylated by Plk1 at Ser-52 of the transactivating (TA) domain. Plk1 decreased the protein stability of TAp63 by its phosphorylation and suppressed TAp63-induced cell death. Furthermore, Plk1 knockdown in p53-mutated liver tumor cells transactivated p53 family downstream effectors, PUMA, p21(Cip1/WAF1) and 14-3-3sigma, and induced apoptotic cell death. Double knockdown of Plk1/p63 attenuated Plk1 knockdown-induced apoptotic cell death and transactivation. Intriguingly, both Plk1 and p63 are highly expressed in the side population (SP) fraction of liver tumor cells compared to non-SP fraction cells, suggesting the significance of Plk1/TAp63 in the control of cell death in tumor-initiating SP fraction cells. Thus, Plk1 controls TAp63 by its phosphorylation and regulates apoptotic cell death in liver tumor cells. Plk1/TAp63 may be a suitable candidate as a molecular target of liver tumor treatments.
Abstract: Tumor suppressor p53 plays a pivotal role in the regulation of cell fate determination in response to a variety of cellular stress including carbon source depletion. In this study, we found that cAMP-responsive element-binding protein (CREB) collaborates with AMP-activated protein kinase alpha (AMPKalpha) to regulate the transcription of p53. Luciferase reporter assays showed that the genomic fragment spanning from -531 to -239 of human p53 gene is required for the transactivation of p53 in response to glucose deprivation. Within this region, we found out a putative CREB-binding site. siRNA-mediated knockdown of CREB resulted in a significant inhibition of the up-regulation of p53 and apoptosis under glucose deprivation. Consistent with these observations, glucose deprivation induced the transcription of p53 and CREB. Additionally, glucose deprivation led to an efficient recruitment of CREB onto the promoter region of p53 gene carrying the canonical CREB-binding site, indicating that CREB has an ability to bind to the promoter region of p53 gene and transactivate p53. Furthermore, the amounts of CREB/phospo-AMPKalpha complex increased in response to glucose deprivation. Taken together, our present findings suggest that p53 is transcriptionally regulated by CREB/phospho-AMPKalpha complex and thereby contributing to the induction of apoptosis under carbon source depletion.
Abstract: Accumulating evidence indicates that TBP (TATA-binding protein)-like protein (TLP) contributes to the regulation of stress-mediated cell cycle checkpoint and apoptotic pathways, although its physiological target genes have remained elusive. In the present study, we have demonstrated that human TAp63 is one of the direct transcriptional target genes of TLP. Enforced expression of TLP results in the transcriptional induction of the endogenous TAp63, but not of the other p53 family members such as TAp73 and p53. Consistent with these results, small interference RNA-mediated knockdown led to a significant down-regulation of the endogenous TAp63. Luciferase reporter assay and chromatin immunoprecipitation analysis revealed that the genomic region located at positions -487 to -29, where +1 represents the transcriptional initiation site of TAp63, is required for TLP-dependent transcriptional activation of TAp63 and also TLP is efficiently recruited onto this region. Additionally, cells treated with anti-cancer drug etoposide underwent apoptosis in association with the transcriptional enhancement of TAp63 in a p53-independent manner, and the knockdown of the endogenous TLP reduced etoposide-induced apoptosis through repression of TAp63 expression. Taken together, our present study identifies a TLP-TAp63 pathway that is further implicated in stress-induced apoptosis.
Abstract: p63 is expressed as multiple variants including TA and DeltaN forms. Since p63-deficient mice displayed profound defects of stratified epithelia, p63 is an essential transcription factor required for epidermal morphogenesis. However, precise molecular mechanisms behind contribution of p63 to normal skin formation and healing skin wounds remained unclear. In this study, we demonstrated that DeltaNp63/BMP-7 signaling pathway modulates wound healing process through the regulation of extracellular matrix protein matrilin-2. Knocking down of DeltaNp63 in human keratinocyte HaCaT cells led to a significant reduction of matrilin-2. Intriguingly, BMP-7 which is one of DeltaNp63-target gene products, induced matrilin-2 and attenuated inhibitory effect of siRNA against DeltaNp63 on matrilin-2. Furthermore, a remarkable cell migration in response to wounding took place in DeltaNp63- or matrilin-2-knocked down cells. Taken together, our present findings indicate that DeltaNp63/BMP-7 signaling pathway modulates wound healing process through the regulation of matrilin-2.
Abstract: NFBD1/MDC1 is a large nuclear protein with an anti-apoptotic potential which participates in DNA damage response. Recently, we have demonstrated that NFBD1 has an inhibitory effect on pro-apoptotic p53 and DNA damage-induced transcriptional repression of NFBD1 plays an important role in p53-dependent apoptotic response. In this study, we have found that NFBD1 promoter region contains canonical Sp1-, STAT-1- and NF-Y-binding sites and finally we have identified Sp1 as a transcriptional activator for NFBD1. The 5'-RACE and bioinformatic analyses revealed that NFBD1 encodes at least four transcriptional variants arising from distinct transcriptional start sites. Luciferase reporter assays using a series of NFBD1 promoter deletion mutants demonstrated that the proximal Sp1-binding site is required for the transcriptional activation of NFBD1. Indeed, the endogenous Sp1 was recruited onto the proximal Sp1-binding site as examined by chromatin immunoprecipitation (ChIP) assay and siRNA-mediated knockdown of the endogenous Sp1 in HeLa cells reduced the expression levels of NFBD1, which renders cells sensitive to adriamycin (ADR). In support of this notion, mithramycin A (MA, Sp1 inhibitor) treatment resulted in a significant down-regulation of NFBD1. Taken together, our present findings suggest that Sp1-mediated transcriptional regulation of NFBD1 plays an important role in the regulation of DNA damage response.
Abstract: Deletion of the distal region of chromosome 1 frequently occurs in a variety of human cancers, including aggressive neuroblastoma. Previously, we have identified a 500-kb homozygously deleted region at chromosome 1p36.2 harboring at least six genes in a neuroblastoma-derived cell line NB1/C201. Among them, only KIF1Bbeta, a member of the kinesin superfamily proteins, induced apoptotic cell death. These results prompted us to address whether KIF1Bbeta could be a tumor suppressor gene mapped to chromosome 1p36 in neuroblastoma. Hemizygous deletion of KIF1Bbeta in primary neuroblastomas was significantly correlated with advanced stages (p = 0.0013) and MYCN amplification (p < 0.001), whereas the mutation rate of the KIF1Bbeta gene was infrequent. Although KIF1Bbeta allelic loss was significantly associated with a decrease in KIF1Bbeta mRNA levels, its promoter region was not hypermethylated. Additionally, expression of KIF1Bbeta was markedly down-regulated in advanced stages of tumors (p < 0.001). Enforced expression of KIF1Bbeta resulted in an induction of apoptotic cell death in association with an increase in the number of cells entered into the G2/M phase of the cell cycle, whereas its knockdown by either short interfering RNA or by a genetic suppressor element led to an accelerated cell proliferation or enhanced tumor formation in nude mice, respectively. Furthermore, we demonstrated that the rod region unique to KIF1Bbeta is critical for the induction of apoptotic cell death in a p53-independent manner. Thus, KIF1Bbeta may act as a haploinsufficient tumor suppressor, and its allelic loss may be involved in the pathogenesis of neuroblastoma and other cancers.
Abstract: Although it has been well documented that loss of human chromosome 11q is frequently observed in primary neuroblastomas, the smallest region of overlap (SRO) has not yet been precisely identified. Previously, we performed array-comparative genomic hybridization (array-CGH) analysis for 236 primary neuroblastomas to search for genomic aberrations with high-resolution. In our study, we have identified the SRO of deletion (10-Mb or less) at 11q23. Within this region, there exists a TSLC1/IGSF4/CADM1 gene (Tumor suppressor in lung cancer 1/Immunoglobulin superfamily 4/Cell adhesion molecule 1), which has been identified as a putative tumor suppressor gene for lung and some other cancers. Consistent with previous observations, we have found that 35% of primary neuroblastomas harbor loss of heterozygosity (LOH) on TSLC1 locus. In contrast to other cancers, we could not detect the hypermethylation in its promoter region in primary neuroblastomas as well as neuroblastoma-derived cell lines. The clinicopathological analysis demonstrated that TSLC1 expression levels significantly correlate with stage, Shimada's pathological classification, MYCN amplification status, TrkA expression levels and DNA index in primary neuroblastomas. The immunohistochemical analysis showed that TSLC1 is remarkably reduced in unfavorable neuroblastomas. Furthermore, decreased expression levels of TSLC1 were significantly associated with a poor prognosis in 108 patients with neuroblastoma. Additionally, TSLC1 reduced cell proliferation in human neuroblastoma SH-SY5Y cells. Collectively, our present findings suggest that TSLC1 acts as a candidate tumor suppressor gene for neuroblastoma.
Abstract: Since p63-deficient mice display severe defects in formation of epidermis, p63 has been considered to be a multi-isoform p53 family member essential for epidermal development. However, it is still unclear how p63 could contribute to keratinocyte differentiation. In the present study, we have found that TAp63alpha is induced in association with the upregulation and a secretion of growth differentiation factor 15 (GDF15) during the keratinocyte differentiation of HaCaT cells bearing p53 mutation. Short interference RNA-mediated knockdown of the endogenous TAp63 resulted in a remarkable reduction of GDF15. Luciferase reporter assay and reverse transcription-PCR analysis demonstrated that enforced expression of TAp63alpha significantly increases the luciferase activity driven by GDF15 promoter and the expression of GDF15. Consistent with these results, the proximal p53/p63-binding site within the GDF15 promoter region was required for the TAp63alpha-mediated transcriptional activation of GDF15, and TAp63alpha was recruited onto this site. Furthermore, siRNA-mediated knockdown of the endogenous GDF15 permitted cell growth and inhibited the expression of the differentiation markers such as keratin 10 and involucrin in response to differentiation stimuli. Taken together, our present results provide a novel insight into understanding the molecular mechanisms behind TAp63alpha-mediated keratinocyte differentiation.
Abstract: TBP-related factor 2 (TRF2), one of the TBP family proteins, is involved in various cellular functions through its transcription stimulation activity. We previously reported that TRF2 is involved in reduction of wee1 mRNA in genotoxin-treated chicken cells. In this study, we investigated the role of TRF2 in wee1 gene expression. It was found that wee1 mRNA was decreased in hydroxyurea-treated NIH3T3 cells. Mouse wee1 promoter activity was repressed by TRF2 in mouse and chicken cells. Chromatin immunoprecipitation and plasmid immunoprecipitation analyses revealed that TRF2 is recruited to the wee1 promoter in accordance with the transcriptional repression. A mutant TRF2 that lacks TFIIA-binding capacity lost its repressive function. This mutant was less recruited to the wee1 promoter than was the wild-type one, and provided a decline in promoter-recruited TFIIA. Data in this study suggest that transcription repressive activity of TRF2 to wee1 promoter needs association with the promoter and TFIIA.
Abstract: TBP (TATA-binding protein)-related factor 2 (TRF2) regulates transcription during a nuber of cellular processes. We previously demonstrated that it is localized in the cytoplasm and is translocated to the nucleus by DNA-damaging agents. However, the cytoplasmic localization of TRF2 is controversial. In this study, we reconfirmed its cytoplasmic localization in various ways and examined its nuclear migration. Stresses such as heat shock, redox agents, heavy metals, and osmotic shock did not affect localization whereas genotoxins such as methyl methanesulfonate (MMS), cisplatin, etoposide, and hydroxyurea caused it to migrate to the nucleus. Adriamycin, mitomycin C and gamma-rays had no obvious effect. We determined optimal conditions for the nuclear migration. The proportions of cells with nuclei enriched for TRF2 were 25-60% and 5-10% for stressed cells and control cells, respectively. Nuclear translocation was observed after 1 h, 4 h and 12 h for cisplatin, etoposide and MMS and hydroxyurea, respectively. The association of TRF2 with the chromatin and promoter region of the proliferating cell nuclear antigen (PCNA) gene, a putative target of TRF2, was increased by MMS treatment. Thus TRF2 may be involved in genotoxin-induced transcriptional regulation.
