Elongation of the developing spinal cord is driven by Oct4-type transcription factor-mediated regulation of retinoic acid signaling in zebrafish embryos.

BACKGROUND: Elongation of the spinal cord is dependent on neural development from neuromesodermal progenitors in the tail bud. We previously showed the involvement of the Oct4-type gene, pou5f3, in this process in zebrafish mainly by dominant-interference gene induction, but, to compensate for the limitation of this transgene approach, mutant analysis was indispensable. pou5f3 involvement in the signaling pathways was another unsolved question. RESULTS: We examined the phenotypes of pou5f3 mutants and the effects of Pou5f3 activation by the tamoxifen-ERT2 system in the posterior neural tube, together confirming the involvement of pou5f3. The reporter assays using P19 cells implicated tail bud-related transcription factors in pou5f3 expression. Regulation of tail bud development by retinoic acid (RA) signaling was confirmed by treatment of embryos with RA and the synthesis inhibitor, and in vitro reporter assays further showed that RA signaling regulated pou5f3 expression. Importantly, the expression of the RA degradation enzyme gene, cyp26a1, was down-regulated in embryos with disrupted pou5f3 activity. CONCLUSIONS: The involvement of pou5f3 in spinal cord extension was supported by using mutants and the gain-of-function approach. Our findings further suggest that pou5f3 regulates the RA level, contributing to neurogenesis in the posterior neural tube.

Involvement of an Oct4-related PouV gene, pou5f3/pou2, in neurogenesis in the early neural plate of zebrafish embryos.

In early vertebrate embryos, the dorsal ectoderm is induced by the axial mesendoderm to form the neural plate, which is given competence to form neural cells by soxB1 genes. Subsequently, neurogenesis proceeds in proneural clusters that are generated by a gene network involving proneural genes and Notch signaling. However, what occurs between early neural induction and the later initiation of neurogenesis has not been fully revealed. In the present study, we demonstrated that during gastrulation, the expression of the Oct4-related PouV gene pou5f3 (also called pou2), which is widely observed at earlier stages, was rapidly localized to an array of isolated spotted domains, each of which coincided with individual proneural clusters. Two-color in situ hybridization confirmed that each pou5f3-expressing domain included a proneural cluster. Further analysis demonstrated that anterior pou5f3 domains straddled the boundaries between rhombomere 1 (r1) and r2, whereas posterior domains were included in r4. The effects of forced expression of an inducible negative dominant-interfering pou5f3 gene suggested that pou5f3 activated early proneural genes, such as neurog1 and ebf2, and also soxB1, but repressed the late proneural genes atoh1a and ascl1b. Furthermore, pou5f3 was considered to repress her4.1, a Notch-dependent Hairy/E(spl) gene involved in lateral inhibition in proneural clusters. These results suggest that pou5f3 promotes early neurogenesis in proneural clusters, but negatively regulates later neurogenesis. Suppression of pou5f3 also altered the expression of other her genes, including her3, her5, and her9, further supporting a role for pou5f3 in neurogenesis. In vitro reporter assays in P19 cells showed that pou5f3 was repressed by neurog1, but activated by Notch signaling. These findings together demonstrate the importance of the pou5f3-mediated gene regulatory network in neural development in vertebrate embryos.

ARID3A regulates autophagy related gene BECN1 expression and inhibits proliferation of osteosarcoma cells.

Osteosarcoma (OS) is the most common primary malignant bone tumor which has unclear pathobiology. Hence, enlightening the exact molecular mechanism underlying osteosarcoma progression is crucial for developing new treatment strategies. One member of the ARID family of DNA binding proteins is ARID3A that is implicated in osteosarcoma pathogenesis. ARID3A could bind E2F1 and regulate the transcription of E2F1 targets. At the same time, BECN1 is a well-characterized autophagy regulator gene that is a direct target of E2F1. The present study aimed to investigate the effect of ARID3A on the expression of BECN1 in osteosarcoma cells. First, we determined gene expression levels of ARID3A, BECN1, and E2F1 in U-2 OS by qPCR and confirmed with online datasets from GEO database. In addition, the prognostic value of these genes was also evaluated from Kaplan-Meier plotter database. Next, ARID3A was overexpressed and silenced in order to investigate the effect of ARID3A on BECN1 expression and proliferation of U-2 OS cells. Our results demonstrated that BECN1 was negatively correlated with E2F1 and positively correlated with ARID3A based on initial expression and prognostic effect in OS. Overexpression of ARID3A upregulated BECN1 while silenced ARID3A downregulated BECN1 expression in U-2 OS cells. Additionally, silencing of ARID3A promoted colony formation and proliferation, whereas overexpression of ARID3A suppressed colony formation and proliferation of U-2 OS cells. Taken together, these results indicate that ARID3A could function as tumor suppressor and affect the expression level of BECN1 in U-2 OS cells.

Involvement of Oct4-type transcription factor Pou5f3 in posterior spinal cord formation in zebrafish embryos.

In vertebrate embryogenesis, elongation of the posterior body is driven by de novo production of the axial and paraxial mesoderm as well as the neural tube at the posterior end. This process is presumed to depend on the stem cell-like population in the tail bud region, but the details of the gene regulatory network involved are unknown. Previous studies suggested the involvement of pou5f3, an Oct4-type POU gene in zebrafish, in axial elongation. In the present study, we first found that pou5f3 is expressed mainly in the dorsal region of the tail bud immediately after gastrulation, and that this expression is restricted to the posterior-most region of the elongating neural tube during somitogenesis. This pou5f3 expression was complementary to the broad expression of sox3 in the neural tube, and formed a sharp boundary with specific expression of tbxta (orthologue of mammalian T/Brachyury) in the tail bud, implicating pou5f3 in the specification of tail bud-derived cells toward neural differentiation in the spinal cord. When pou5f3 was functionally impaired after gastrulation by induction of a dominant-interfering pou5f3 mutant gene (en-pou5f3), trunk and tail elongation were markedly disturbed at distinct positions along the axis depending on the stage. This finding showed involvement of pou5f3 in de novo generation of the body from the tail bud. Conditional functional abrogation also showed that pou5f3 downregulates mesoderm-forming genes but promotes neural development by activating neurogenesis genes around the tail bud. These results suggest that pou5f3 is involved in formation of the posterior spinal cord.

Long noncoding RNA ERICD interacts with ARID3A via E2F1 and regulates migration and proliferation of osteosarcoma cells.

Long noncoding RNA (lncRNA) dysregulation is known to be taking part in majority of cancers, including osteosarcoma. In one of our previous studies, we showed that lncRNA MEG3 is being regulated by microRNA-664a (miR-664a) suppresses the migratory potential of osteosarcoma cells (U-2OS). We now report a novel lncRNA, namely, ERICD, which is linked to the transcription factor AT-rich interaction domain 3A (ARID3A) in U-2OS cells. We show that ARID3A binds to ERICD and indirectly interacts with each other via the E2F transcription factor 1 (E2F1). Furthermore, small interfering RNA (siRNA)-mediated knockdown of ERICD inhibited cell migration, formation of colonies, and proliferation in U-2OS cells. Overexpression of ARID3A inhibited cell migration, colony formation, and proliferation, whereas siRNA-mediated knockdown of ARID3A promoted cell migration, colony formation, and proliferation. Our findings indicate that ARID3A and lncRNA ERICD have plausible tumor suppressive and oncogenic functions, respectively, in osteosarcoma. Our data demonstrate the converse interaction between ARID3A and lncRNA ERICD that target DNA-binding proteins and dysregulation of their expression through E2F1 augments osteosarcoma progression. The cell rescue experiment also indicated E2F1 to be involved in the regulation of ARID3A and ERICD.

miR-222 regulates proliferation of primary mouse hepatocytes in vitro.

It is well known that hepatocytes regenerate after liver injury, although it is difficult to reproduce this phenomenon in vitro. The goal of this research was to determine the factors that stimulate proliferation of primary mouse hepatocytes (PMHs) in vitro. We first tested knockdown (KD) of tumor protein 53 (p53) alone as well as partial hepatectomy (PH, performed 72 h prior to PMHs preparation) alone. However, neither intervention stimulated hepatocyte proliferation during the 72-h observation period in vitro. We then tested the combination of p53 KD with PH and found that these interventions together stimulated cell proliferation in vitro. Under these latter conditions we analyzed gene expression of these cells by mRNA sequencing (RNA-seq) and microRNA sequencing (miRNA-seq). TargetScan analysis, which determines the relationship between microRNAs and gene expression, found a relationship between downregulated mmu-mir-222 (miR-222) and upregulated genes such as mitogen-activated protein kinase kinase kinase 2 (Map3k2). To confirm this relationship, we performed miR-222 KD and overexpression (OE) and observed the expected changes in target gene expression. Furthermore, the finding that miR-222 KD or OE stimulates or suppresses, respectively, hepatocyte proliferation is well explained by the association between miR-222 and its target genes, which stimulate growth. Our results suggest that miR-222 is one of the key factors regulating PMH proliferation in vitro.

In vitro analysis of the transcriptional regulatory mechanism of zebrafish pou5f3.

Zebrafish pou5f3 (previously named pou2), a close homologue of mouse Oct4, encodes a PouV-family transcription factor. pou5f3 has been implicated in diverse aspects of developmental regulation during embryogenesis. In the present study, we addressed the molecular function of Pou5f3 as a transcriptional regulator and the mechanism by which pou5f3 expression is transcriptionally regulated. We examined the influence of effector genes on the expression of the luciferase gene under the control of the upstream 2.1-kb regulatory DNA of pou5f3 (Luc-2.2) in HEK293T and P19 cells. We first confirmed that Pou5f3 functions as a transcriptional activator both in cultured cells and embryos, which confirmed autoregulation of pou5f3 in embryos. It was further shown that Luc-2.2 was activated synergistically by pou5f3 and sox3, which is similar to the co-operative activity of Oct4 and Sox2 in mice, although synergy between pou5f3 and sox2 was less obvious in this zebrafish system. The effects of pou5f3 deletion constructs on the regulation of Luc-2.2 expression revealed different roles for the three subregions of the N-terminal region in Pou5f3 in terms of its regulatory functions and co-operativity with Sox3. Electrophoretic mobility shift assays confirmed that Pou5f3 and Sox3 proteins specifically bind to adjacent sites in the 2.1-kb DNA and that there is an interaction between the two proteins. The synergy with sox3 was unique to pou5f3-the other POU factor genes examined did not show such synergy in Luc-2.2 regulation. Finally, functional interaction was observed between pou5f3 and sox3 in embryos in terms of the regulation of dorsoventral patterning and convergent extension movement. These findings together demonstrate co-operative functions of pou5f3 and sox3, which are frequently coexpressed in early embryos, in the regulation of early development.

Extended Culture Conditions for Multipotent Bone Marrow-Derived Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) offer a promising source of cells for musculoskeletal regeneration because of their potential to differentiate into bone, cartilage and fat. However, their proliferation and multilineage differentiation potential decreases with aging or increased time in in vitro culture. To determine culture conditions capable of enabling maintenance of MSCs for extended periods of time, human bone marrow-derived MSCs (BM-MSCs) were cultured in growth medium containing various combinations of growth factors and small chemical compounds. Upon reaching confluence, MSCs were subcultured continuously and then tested for differentiation capacity. After screening various growth factors and small chemical compounds, we found a combination capable of maintaining the proliferation potential of BM-MSCs obtained from a 19-year-old donor (young MSCs) up to passage 13 (P13). In contrast, unsupplemented MSCs reached senescence at P10. Total population doublings of control (P10) and supplemented MSCs (P12) were estimated at 20.4 and 42, respectively. Young MSCs cultured with supplements maintained osteogenic, adipogenic and chondrogenic differentiation capacities at P12 as confirmed by expression of lineage-specific differentiation markers. Furthermore, the supplementation of to BM-MSCs obtained from 65- and 79-year-old donors (aged MSCs) also continued to proliferate until P12, and maintained osteogenic and adipogenic differentiation capacity until P7 and P8, respectively, whereas, unsupplemented aged MSCs stopped proliferating at P8. These results indicate that our extended culture conditions maintained the proliferative capacity of young MSCs while retaining their multipotent differentiation potential, and improved both proliferation and differentiation of aged MSCs.

Bmal1 is an essential regulator for circadian cytosolic Ca²âº rhythms in suprachiasmatic nucleus neurons.

The hypothalamic suprachiasmatic nucleus (SCN) plays a pivotal role in the mammalian circadian clock system. Bmal1 is a clock gene that drives transcriptional-translational feedback loops (TTFLs) for itself and other genes, and is expressed in nearly all SCN neurons. Despite strong evidence that Bmal1-null mutant mice display arrhythmic behavior under constant darkness, the function of Bmal1 in neuronal activity is unknown. Recently, periodic changes in the levels of intracellular signaling messengers, such as cytosolic Ca(2+) and cAMP, were suggested to regulate TTFLs. However, the opposite aspect of how clock gene TTFLs regulate cytosolic signaling remains unclear. To investigate intracellular Ca(2+) dynamics under Bmal1 perturbations, we cotransfected some SCN neurons with yellow cameleon together with wild-type or dominant-negative Bmal1 using a gene-gun applied for mouse organotypic cultures. Immunofluorescence staining for a tag protein linked to BMAL1 showed nuclear expression of wild-type BMAL1 and its degradation within 1 week after transfection in SCN neurons. However, dominant-negative BMAL1 did not translocate into the nucleus and the cytosolic signals persisted beyond 1 week. Consistently, circadian Ca(2+) rhythms in SCN neurons were inhibited for longer periods by dominant-negative Bmal1 overexpression. Furthermore, SCN neurons transfected with a Bmal1 shRNA lengthened, whereas those overexpressing wild-type Bmal1 shortened, the periods of Ca(2+) rhythms, with a significant reduction in their amplitude. BMAL1 expression was intact in the majority of neighboring neurons in organotypic cultures. Therefore, we conclude that proper intrinsic Bmal1 expression, but not passive signaling via cell-to-cell interactions, is the determinant of circadian Ca(2+) rhythms in SCN neurons.

Critical role of ARID3B in the expression of pro-apoptotic p53-target genes and apoptosis.

ARID3A and ARID3B are transcriptional targets of p53. Recently, it has been reported that ARID3A plays a critical role in the transcriptional activation of pro-arrest p21 in response to DNA damage. However, the role of ARID3B in the p53 regulatory pathway remains poorly understood. Here we show that ARID3A and ARID3B specifically bind to putative ARID3-binding sites in p53 target genes in vitro and in vivo. ARID3B and, to a lesser extent, ARID3A silencing blocked transcriptional activation of pro-apoptotic p53 target genes, such as PUMA, PIG3, and p53. Furthermore, ectopic ARID3B, to a lesser extent, ARID3A expression activated the pro-apoptotic gene expression, and only ARID3B induced apoptosis. Finally, ARID3B but not ARID3A silencing blocked apoptosis induction following DNA damage. These results indicated that, although ARID3B and ARID3A share overlapping functions, ARID3B play a key role in the expression of pro-apoptotic p53-target genes and apoptosis.

Identification of two nickel ion-induced genes, NCI16 and PcGST1, in Paramecium caudatum.

Here, we describe the isolation of two nickel-induced genes in Paramecium caudatum, NCI16 and PcGST1, by subtractive hybridization. NCI16 encoded a predicted four-transmembrane domain protein (∼16 kDa) of unknown function, and PcGST1 encoded glutathione S-transferase (GST; ∼25 kDa) with GST and glutathione peroxidase (GPx) activities. Exposing cells to cobalt chloride also caused the moderate upregulation of NCI16 and PcGST1 mRNAs. Both nickel sulfate and cobalt chloride dose dependently induced NCI16 and PcGST1 mRNAs, but with different profiles. Nickel treatment caused a continuous increase in PcGST1 and NCI16 mRNA levels for up to 3 and 6 days, respectively, and a notable increase in H2O2 concentrations in P. caudatum. NCI16 expression was significantly enhanced by incubating cells with H2O2, implying that NCI16 induction in the presence of nickel ions is caused by reactive oxygen species (ROS). On the other hand, PcGST1 was highly induced by the antioxidant tert-butylhydroquinone (tBHQ) but not by H2O2, suggesting that different mechanisms mediate the induction of NCI16 and PcGST1. We introduced a luciferase reporter vector with an ∼0.42-kb putative PcGST1 promoter into cells and then exposed the transformants to nickel sulfate. This resulted in significant luciferase upregulation, indicating that the putative PcGST1 promoter contains a nickel-responsive element. Our nickel-inducible system also may be applicable to the efficient expression of proteins that are toxic to host cells or require temporal control.

Shared Mechanisms in Pparγ1sv and Pparγ2 Expression in 3T3-L1 Cells: Studies on Epigenetic and Positive Feedback Regulation of Pparγ during Adipogenesis.

We have previously reported the identification of a novel splicing variant of the mouse peroxisome proliferator-activated receptor-γ (Pparγ), referred to as Pparγ1sv. This variant, encoding the PPARγ1 protein, is abundantly and ubiquitously expressed, playing a crucial role in adipogenesis. Pparγ1sv possesses a unique promoter and 5' untranslated region (5'UTR), distinct from those of the canonical mouse Pparγ1 and Pparγ2 mRNAs. We observed a significant increase in DNA methylation at two CpG sites within the proximal promoter region (-733 to -76) of Pparγ1sv during adipocyte differentiation. Concurrently, chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) using antibodies against H3K4me3 and H3K27ac indicated marked elevations in both methylation and acetylation of histone H3, while the repressive histone mark H3K9me2 significantly decreased, at the transcription start sites of both Pparγ1sv and Pparγ2 following differentiation. Knocking down Pparγ1sv using specific siRNA also led to a decrease in Pparγ2 mRNA and PPARγ2 protein levels; conversely, knocking down Pparγ2 resulted in reduced Pparγ1sv mRNA and PPARγ1 protein levels, suggesting synergistic transcriptional regulation of Pparγ1sv and Pparγ2 during adipogenesis. Furthermore, our experiments utilizing the CRISPR-Cas9 system identified crucial PPARγ-binding sites within the Pparγ gene locus, underscoring their significance in adipogenesis. Based on these findings, we propose a model of positive feedback regulation for Pparγ1sv and Pparγ2 expression during the adipocyte differentiation process in 3T3-L1 cells.

Droplet manipulation by an external electric field for crystalline film growth.

Combining droplet manipulation by the application of an electric field with inkjet printing is proposed as a unique technique to control the surface wettability of substrates for solution-processed organic field-effect transistors (FETs). With the use of this technique, uniform thin films of 2,7-dioctyl[1]benzothieno[2,3,-b][1]benzothiopene (C8-BTBT) could be fabricated on the channels of FET substrates without self-assembled monolayer treatment. High-speed camera observation revealed that the crystals formed at the solid/liquid interface. The coverage of the crystals on the channels depended on the ac frequency of the external electric field applied during film formation, leading to a wide variation in the carrier transport of the films. The highest hole mobility of 0.03 cm(2) V(-1) s(-1) was obtained when the coverage was maximized with an ac frequency of 1 kHz.

Integrated Bone Formation Through In Vivo Endochondral Ossification Using Mesenchymal Stem Cells.

Conventional bone regeneration therapy using mesenchymal stem cells (MSCs) is difficult to apply to bone defects larger than the critical size because it does not have a mechanism to induce angiogenesis. Implanting artificial cartilage tissue fabricated from MSCs induces angiogenesis and bone formation in vivo via endochondral ossification (ECO). Therefore, this ECO-mediated approach may be a promising bone regeneration therapy in the future. An important aspect of the clinical application of this ECO-mediated approach is establishing a protocol for preparing enough cartilage to be implanted to repair the bone defect. It is especially not practical to design a single mass of grafted cartilage of a size that conforms to the shape of the actual bone defect. Therefore, the cartilage to be transplanted must have the property of forming bone integrally when multiple pieces are implanted. Hydrogels may be an attractive tool for scaling up tissue-engineered grafts for endochondral ossification to meet clinical requirements. Although many naturally derived hydrogels support MSC cartilage formation in vitro and ECO in vivo, the optimal scaffold material to meet the needs of clinical applications has yet to be determined. Hyaluronic acid (HA) is a crucial component of the cartilage extracellular matrix and is a biodegradable and biocompatible polysaccharide. Here, we show that HA hydrogels have excellent properties to support in vitro differentiation of MSC-based cartilage tissue and promote endochondral bone formation in vivo.

Temporal inhibition of the electron transport chain attenuates stress-induced cellular senescence by prolonged disturbance of proteostasis in human fibroblasts.

We previously developed a stress-induced premature senescence (SIPS) model in which normal human fibroblast MRC-5 cells were treated with either the proteasome inhibitor MG132 or the vacuolar-type ATPase inhibitor bafilomycin A1 (BAFA1). To clarify the involvement of mitochondrial function in our SIPS model, MRC-5 cells were treated with MG132 or BAFA1 along with an inhibitor targeting either the electron transport chain complex I or complex III, or with a mitochondrial uncoupler. SIPS induced by MG132 or BAFA1 was significantly attenuated by short-term co-treatment with the complex III inhibitor, antimycin A (AA), but not the complex I inhibitor, rotenone or the mitochondrial uncoupler, carbonyl cyanide 3-chlorophenylhydrazone. By co-treatment with AA, mitochondrial and intracellular reactive oxygen species levels, accumulation of protein aggregates and mitochondrial unfolded protein responses (UPRmt ) were remarkably suppressed. Furthermore, AA co-treatment suppressed the hyperpolarization of the mitochondrial membrane and the induction of mitophagy observed in MG132-treated cells and enhanced mitochondrial biogenesis. These findings provide evidence that the temporal inhibition of mitochondrial respiration exerts protective effects against the progression of premature senescence caused by impaired proteostasis.

Hyaluronic acid hydrogels support to generate integrated bone formation through endochondral ossification in vivo using mesenchymal stem cells.

Engineered cartilage tissue from differentiated mesenchymal stem cells (MSCs) can generate bone in vivo through endochondral ossification (ECO). This ECO-mediated approach has the potential to circumvent the severe problems associated with conventional MSC-based bone tissue engineering techniques that lack mechanisms to induce angiogenesis. Hyaluronic acid (HA) is a key component in the cartilage extracellular matrix. However, the ECO-supporting properties of HA remain largely unclear. This study aimed to compare the ability of HA and collagen hydrogels to support in vitro differentiation of MSC-based hypertrophic cartilage tissues and to promote endochondral bone formation in vivo. Following the chondrogenic and hypertrophic differentiation in vitro, both HA and collagen constructs accumulated sulfated glycosaminoglycan (sGAG) and type 1, type II, and type X collagen. However, HA hydrogels exhibited a more uniform distribution of sGAG, type 1 collagen, type X collagen, and osteocalcin proteins; in addition, the cells embedded in the hydrogels had more rounded cell morphologies than those in the collagen constructs. At week 5 of in vitro culture, two to three constructs were implanted into a subcutaneous pocket in nude mice and harvested after 4 and 8 weeks. Both HA and collagen constructs promoted endochondral bone formation with vascularization and bone marrow development; however, the HA constructs fused to form integrated bone tissues and the bone marrow developed along the space between the two adhered grafts in all implanted pockets (n = 5). In the collagen constructs, the integration was observed in 40% of the pockets (n = 5). Microcomputer CT analysis revealed that the bone volume of HA constructs was larger than that of collagen constructs. In conclusion, compared to collagen hydrogels, HA hydrogels had superior potential to generate integrated bone with vascularization and bone marrow development. This study provides valuable insights for applying ECO-mediated bone tissue engineering approaches for the repair of critical-sized bone defects.

ARID3A and ARID3B exert direct regulatory control over the long non-coding RNAs (lncRNAs) MALAT1 and NORAD within the context of non-small cell lung cancer (NSCLC).

Lung cancer, known for its high mortality rates and poor prognosis, remains one of the most prevalent cancer types. Early detection and effective treatment methods are crucial for improving survival rates. Non-small cell lung cancer (NSCLC) accounts for approximately 85 % of all lung cancer cases. Long non-coding RNAs (lncRNAs), which play vital roles in various biological processes, have been implicated in the development of cancer and can impact key therapeutic targets in different cancer types. In NSCLC, the dysregulation of specific lncRNAs, such as MALAT1 and NORAD, has been associated with neoplastic initiation, progression, metastasis, tumor angiogenesis, chemoresistance, and genomic instability. Both MALAT1 and NORAD directly regulate the expression of the transcription factor E2F1, thereby influencing cell cycle progression. Additionally, MALAT1 has been reported to affect the expression of p53 target genes, leading to cell cycle progression through the repression of p53 promoter activity. NORAD, on the other hand, is indirectly regulated by p53. The AT-rich interaction domain (ARID) family of DNA-binding proteins, particularly ARID3A and ARID3B, are involved in various biological processes such as cell proliferation, differentiation, and development. They also play significant roles in E2F-dependent transcription and are transcriptional targets of p53. The intricate balance between promoting cellular proliferation through the pRB-E2F pathway and inducing growth arrest through the p53 pathway underscores the crucial regulatory role of ARID3A, ARID3B, and their interaction with lncRNAs MALAT1 and NORAD. In this study, we aimed to investigate the potential interactive and functional connections among ARID3A, ARID3B, MALAT1, and NORAD in NSCLC, considering their involvement in the pRB-E2F and p53 pathways. Our findings strongly suggest that ARID3A and ARID3B play a regulatory role in controlling MALAT1 and NORAD in NSCLC. Specifically, our study demonstrates that the activities of MALAT1 and NORAD were markedly increased upon the overexpression of ARID3A and ARID3B. Therefore, we can conclude that ARID3A and ARID3B likely contribute significantly to the oncogenic functions of MALAT1 and NORAD in NSCLC. Consequently, targeting ARID3A and ARID3B could hold promise as a therapeutic approach in NSCLC, given their direct control over the expression of MALAT1 and NORAD.

Cooperation between ARID3A and p53 in the transcriptional activation of p21WAF1 in response to DNA damage.

ARID3A/DRIL1/Bright is a family member of the AT rich interaction domain (ARID) DNA-binding proteins that are involved in diverse biological processes. We have reported that p53 activates ARID3A transcription, and ARID3A overexpression induces G1 arrest. However, the role of ARID3A in the p53 pathway remains unclear. Here, we show that ARID3A cooperates with p53 to transcriptionally activate p21(WAF1), a p53-target gene important for cell-cycle arrest. ARID3A bound to its binding sites in the p21(WAF1) promoter in vivo and in vitro, and induced p21(WAF1) transcription in U2OS cells expressing wild-type p53 but not Saos-2 cells lacking p53. The co-expression of ARID3A with p53 cooperates to activate p21(WAF1) transcription and the stably transfected p21(WAF1) promoter. Mutation of the ARID3A binding sites reduced the p21(WAF1) promoter activity, and siRNA-based ARID3A knockdown suppressed the transcription of p21(WAF1), but not the proapoptotic NOXA and PUMA in response to DNA damage. Furthermore, p53 knockdown decreased ARID3A transcription, and, conversely, ARID3A overexpression and knockdown resulted in an increase or decrease in p53 stability, respectively. These results indicate both cooperative and interdependent roles for ARID3A and p53 in the transcriptional activation of p21(WAF1) in response to DNA damage.

Inability of p53-reactivating compounds Nutlin-3 and RITA to overcome p53 resistance in tumor cells deficient in p53Ser46 phosphorylation.

The p53 tumor suppressor protein plays key roles in protecting cells from tumorigenesis. Phosphorylation of p53 at Ser46 (p53Ser46) is considered to be a crucial modification regulating p53-mediated apoptosis. Because the activity of p53 is impaired in most human cancers, restoration of wild-type p53 (wt-p53) function by its gene transfer or by p53-reactivating small molecules has been extensively investigated. The p53-reactivating compounds Nutlin-3 and RITA activate p53 in the absence of genotoxic stress by antagonizing the action of its negative regulator Mdm2. Although controversial, Nutlin-3 was shown to induce p53-mediated apoptosis in a manner independent of p53 phosphorylation. Recently, RITA was shown to induce apoptosis by promoting p53Ser46 phosphorylation. Here we examined whether Nutlin-3 or RITA can overcome resistance to p53-mediated apoptosis in p53-resistant tumor cell lines lacking the ability to phosphorylate p53Ser46. We show that Nutlin-3 did not rescue the apoptotic defect of a Ser46 phosphorylation-defective p53 mutant in p53-sensitive tumor cells, and that RITA neither restored p53Ser46 phosphorylation nor induced apoptosis in p53Ser46 phosphorylation-deficient cells retaining wt-p53. Furthermore, treatment with Nutlin-3 or RITA together with adenoviral p53 gene transfer also failed to induce apoptosis in p53Ser46 phosphorylation-deficient cells either expressing or lacking wt-p53. These results indicate that neither Nutlin-3 nor RITA in able to induce p53-mediated apoptosis in the absence of p53Ser46 phosphorylation. Thus, the dysregulation of this phosphorylation in tumor cells may be a critical factor that limits the efficacy of these p53-based cancer therapies.

Contact resistance and megahertz operation of aggressively scaled organic transistors.

Bottom-gate, top-contact organic thin-film transistors (TFTs) with excellent static characteristics (on/off ratio: 10(7) ; intrinsic mobility: 3 cm(2) (V s)(-1) ) and fast unipolar ring oscillators (signal delay as short as 230 ns per stage) are fabricated. The significant contribution of the transfer length to the relation between channel length, contact length, contact resistance, effective mobility, and cutoff frequency of the TFTs is theoretically and experimentally analyzed.