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Cellular senescence provokes a dramatic alteration of chromatin organization and gene expression profile of proinflammatory factors, thereby contributing to various age-related pathologies via the senescence-associated secretory phenotype (SASP). Chromatin organization and global gene expression are maintained through the CCCTC-binding factor (CTCF). However, the molecular mechanism underlying CTCF regulation and its association with SASP gene expression remains to be fully elucidated. A recent study by our team showed that noncoding RNA (ncRNA) derived from normally silenced pericentromeric repetitive sequences directly impair the DNA binding of CTCF. This CTCF disturbance increases the accessibility of chromatin at the loci of SASP genes and caused the transcription of inflammatory factors. This mechanism may promote malignant transformation.

Background: The co-administration of several therapeutic oligonucleotides targeting the same transcript is a beneficial approach. It broadens the target sites for diseases associated with various mutations or splice variants. However, little is known how a combination of antisense oligonucleotides (ASOs), which is one of the major modalities of therapeutic oligonucleotides, affects the potency. In this study, we aimed to elucidate the cocktail-effects of ASOs and the relationship between the target sites and potency of different combinations. Method and Results: We designed 113 ASOs targeting human superoxide dismutase 1 pre-mRNA and found 13 ASOs that had comparable silencing activity in vitro. An analysis of cocktail-effects on the silencing potency of 38 pairs of two ASOs on HeLa cells revealed that 30 pairs had comparable potency to that of two ASOs; on the other hand, eight pairs had reduced potency, indicating a negative impact on the activity. A reduced potency was seen in pairs targeting the same intron, exon-intron combination, or two different introns. The sequence distance of target sites was not the major determinant factor of cocktail-effects. In addition, a cocktail of three ASOs preserving the potency could be designed by avoiding two-ASO pairs, which had a reduced potency. Conclusions: This study revealed that more than half of the combinations retain their potency by paring two ASOs; in contrast, some pairs had a reduced potency. This could not be predicted only by the distance between the target sites.

Achieving regulation of endogenous gene expression in the central nervous system (CNS) with antisense oligonucleotides (ASOs) administered systemically would facilitate the development of ASO-based therapies for neurological diseases. We demonstrate that DNA/RNA heteroduplex oligonucleotides (HDOs) conjugated to cholesterol or α-tocopherol at the 5′ end of the RNA strand reach the CNS after subcutaneous or intravenous administration in mice and rats. The HDOs distribute throughout the brain, spinal cord and peripheral tissues and suppress the expression of four target genes by up to 90% in the CNS, whereas single-stranded ASOs conjugated to cholesterol have limited activity. Gene knockdown was observed in major CNS cell types and was greatest in neurons and microglial cells. Side effects, such as thrombocytopenia and focal brain necrosis, were limited by using subcutaneous delivery or by dividing intravenous injections. By crossing the blood–brain barrier more effectively, cholesterol-conjugated HDOs may overcome the limited efficacy of ASOs targeting the CNS without requiring intrathecal administration. Genes in the rodent brain are knocked down by DNA/RNA heteroduplexes injected intravenously.

Cellular senescence is historically regarded as a tumor suppression mechanism to prevent damaged cells from aberrant proliferation in benign and premalignant tumors. However, recent findings have suggested that senescent cells contribute to tumorigenesis and age‐associated pathologies through the senescence‐associated secretory phenotype (SASP). Therefore, to control age‐associated cancer, it is important to understand the molecular mechanisms of the SASP in the cancer microenvironment. New findings have suggested that the cyclic GMP‐AMP synthase (cGAS)‐stimulator of interferon genes (STING) signaling pathway, a critical indicator of innate immune response, triggers the SASP in response to accumulation of cytoplasmic DNA (cytoplasmic chromatin fragments, mtDNA and cDNA) in senescent cells. Notably, the cGAS‐STING signaling pathway promotes or inhibits tumorigenesis depending on the biological context in vivo, indicating that it may be a potential therapeutic target for cancer. Herein, we review the regulatory machinery and biological function of the SASP via the cGAS‐STING signaling pathway in cancer., This brief review discusses the role of the senescence‐associated secretory phenotype (SASP) and cyclic GMP‐AMP synthase‐stimulator of interferon genes (cGAS‐STING) signaling pathway on cellular senescence in cancer development. We suggest that the cGAS‐STING signaling pathway may be a novel target for induction of SASP.

Cellular senescence caused by oncogenic stimuli is associated with the development of various age-related pathologies through the senescence-associated secretory phenotype (SASP). SASP is mediated by the activation of cytoplasmic nucleic acid sensors. However, the molecular mechanism underlying the accumulation of nucleotide ligands in senescent cells is unclear. In this study, we revealed that the expression of RNaseH2A, which removes ribonucleoside monophosphates (rNMPs) from the genome, is regulated by E2F transcription factors, and it decreases during cellular senescence. Residual rNMPs cause genomic DNA fragmentation and aberrant activation of cytoplasmic nucleic acid sensors, thereby provoking subsequent SASP factor gene expression in senescent cells. In addition, RNaseH2A expression was significantly decreased in aged mouse tissues and cells from individuals with Werner syndrome. Furthermore, RNaseH2A degradation using the auxin-inducible degron system induced the accumulation of nucleotide ligands and induction of certain tumourigenic SASP-like factors, promoting the metastatic properties of colorectal cancer cells. Our results indicate that RNaseH2A downregulation provokes SASP through nucleotide ligand accumulation, which likely contributes to the pathological features of senescent, progeroid, and cancer cells.

Significance During the aging process, senescent cells secrete inflammatory factors, causing various age-related pathologies. Thus, controlling the senescence-associated secretory phenotype (SASP) can tremendously benefit human health. Although SASP seems to be induced by the alteration of chromosomal organization, its underlying mechanism remains unclear. Here, it has been revealed that noncoding RNA (ncRNA) transcribed from pericentromeric repetitive elements impairs the DNA binding of CCCTC-binding factor, resulting in the alteration of chromosomal accessibility and the activation of SASP-like inflammatory genes. Notably, the ncRNA was transferred into surrounding cells via small extracellular vesicles, acting as a tumorigenic SASP factor. Our study highlights a novel mechanism regulating chromatin interaction and inflammatory gene expression in senescence and cancer., Cellular senescence causes a dramatic alteration of chromatin organization and changes the gene expression profile of proinflammatory factors, thereby contributing to various age-related pathologies through the senescence-associated secretory phenotype (SASP). Chromatin organization and global gene expression are maintained by the CCCTC-binding factor (CTCF); however, the molecular mechanism underlying CTCF regulation and its association with SASP gene expression remains unclear. We discovered that noncoding RNA (ncRNA) derived from normally silenced pericentromeric repetitive sequences directly impairs the DNA binding of CTCF. This CTCF disturbance increases the accessibility of chromatin and activates the transcription of SASP-like inflammatory genes, promoting malignant transformation. Notably, pericentromeric ncRNA was transferred into surrounding cells via small extracellular vesicles acting as a tumorigenic SASP factor. Because CTCF blocks the expression of pericentromeric ncRNA in young cells, the down-regulation of CTCF during cellular senescence triggers the up-regulation of this ncRNA and SASP-related inflammatory gene expression. In this study, we show that pericentromeric ncRNA provokes chromosomal alteration by inhibiting CTCF, leading to a SASP-like inflammatory response in a cell-autonomous and non–cell-autonomous manner and thus may contribute to the risk of tumorigenesis during aging.

DNA damage, caused by various oncogenic stresses, can induce cell death or cellular senescence as an important tumor suppressor mechanism. Senescent cells display the features of a senescence-associated secretory phenotype (SASP), secreting inflammatory proteins into surrounding tissues, and contributing to various age-related pathologies. In addition to this inflammatory protein secretion, the release of extracellular vesicles (EVs) is also upregulated in senescent cells. However, the molecular mechanism underlying this phenomenon remains unclear. Here, we show that DNA damage activates the ceramide synthetic pathway, via the downregulation of sphingomyelin synthase 2 (SMS2) and the upregulation of neutral sphingomyelinase 2 (nSMase2), leading to an increase in senescence-associated EV (SA-EV) biogenesis. The EV biogenesis pathway, together with the autophagy-mediated degradation pathway, functions to block apoptosis by removing cytoplasmic DNA fragments derived from chromosomal DNA or bacterial infections. Our data suggest that this SA-EV pathway may play a prominent role in cellular homeostasis, particularly in senescent cells. In summary, DNA damage provokes SA-EV release by activating the ceramide pathway to protect cells from excessive inflammatory responses.

Cellular senescence caused by oncogenic stimuli is related to the development of various age-related diseases via senescence-associated secretory phenotype (SASP). Recent studies have revealed that the SASP is induced via the cGAS-STING (cyclic GMP-AMP synthase linked to stimulator of interferon genes) pathway. However, the molecular mechanism of nucleotide ligand production for these cytoplasmic sensors remains unclear. Here, we discover that the expression of RNaseH2A is regulated by E2F transcription factor and decreases during cellular senescence, thus abnormally incorporated ribonucleoside monophosphates (rNMPs) cause genomic DNA fragmentation, while excess R-loop structures lead to RNA-DNA hybrid accumulation in the nucleoli and cytoplasm of senescent cells. Consequently, both chromosomal DNA fragments and RNA-DNA hybrids induce aberrant activation of the cGAS-STING pathway and SASP-factor gene expression. Furthermore, RNaseH2A reduction was associated with pathological features and poor prognoses in individuals with Werner syndrome and cervical, ovarian and colorectal cancers. Our findings provide new insights into how nucleotide ligand production relates to SASP induction.

Various physiological factors in the gastrointestinal (GI) tract affect the in vivo performance of orally administered dosage forms. In this review article, we summarize the formulation-related interspecies differences (particularly under the fasted state) between humans and laboratory animals. In particular, we discuss the differences in fluid characteristics in the stomach and small intestine, transit of dosage forms in the gut, and drug permeation through the intestinal epithelium after release from dosage forms. Further, we provide several case examples of oral drug formulations that were affected by species-specific differences in GI physiology between laboratory animals and humans that have been discussed at the consortium of Academy of Pharmaceutical Science and Technology, Japan. In these examples, species-specific differences in gut physiological parameters between humans and animals greatly affected the in vivo performance of dosage forms.

Accumulating evidence indicates that the senescence-associated secretory phenotype (SASP) contributes to many aspects of physiology and disease. Thus, controlling the SASP will have tremendous impacts on our health. However, our understanding of SASP regulation is far from complete. Here, we show that cytoplasmic accumulation of nuclear DNA plays key roles in the onset of SASP. Although both DNase2 and TREX1 rapidly remove the cytoplasmic DNA fragments emanating from the nucleus in pre-senescent cells, the expression of these DNases is downregulated in senescent cells, resulting in the cytoplasmic accumulation of nuclear DNA. This causes the aberrant activation of cGAS-STING cytoplasmic DNA sensors, provoking SASP through induction of interferon-β. Notably, the blockage of this pathway prevents SASP in senescent hepatic stellate cells, accompanied by a decline of obesity-associated hepatocellular carcinoma development in mice. These findings provide valuable new insights into the roles and mechanisms of SASP and possibilities for their control., Activation of DNA damage response induces the acquisition of senescence-associated secretory phenotype (SASP) in senescent cells, but precise mechanisms remain unclear. Here, the authors show that the cytoplasmic accumulation of nuclear DNA activated cytoplasmic DNA sensors to provoke SASP.

Tumor cell-induced platelet aggregation facilitates hematogenous metastasis by promoting tumor embolization, preventing immunological assaults and shear stress, and the platelet-releasing growth factors support tumor growth and invasion. Podoplanin, also known as Aggrus, is a type I transmembrane mucin-like glycoprotein and is expressed on wide range of tumor cells. Podoplanin has a role in platelet aggregation and metastasis formation through the binding to its platelet receptor, C-type lectin-like receptor 2 (CLEC-2). The podoplanin research was originally started from the cloning of highly metastatic NL-17 subclone from mouse colon 26 cancer cell line and from the establishment of 8F11 monoclonal antibody (mAb) that could neutralize NL-17-induced platelet aggregation and hematogenous metastasis. Later on, podoplanin was identified as the antigen of 8F11 mAb, and its ectopic expression brought to cells the platelet-aggregating abilities and hematogenous metastasis phenotypes. From the 8F11 mAb recognition epitopes, podoplanin is found to contain tandemly repeated, highly conserved motifs, designated platelet aggregation-stimulating (PLAG) domains. Series of analyses using the cells expressing the mutants and the established neutralizing anti-podoplanin mAbs uncovered that both PLAG3 and PLAG4 domains are associated with the CLEC-2 binding. The neutralizing mAbs targeting PLAG3 or PLAG4 could suppress podoplanin-induced platelet aggregation and hematogenous metastasis through inhibiting the podoplanin-CLEC-2 binding. Therefore, these domains are certainly functional in podoplanin-mediated metastasis through its platelet-aggregating activity. This review summarizes the platelet functions in metastasis formation, the role of platelet aggregation-inducing factor podoplanin in pathological and physiological situations, and the possibility to develop podoplanin-targeting drugs in the future.

Purine nucleosides of 2′-O,4′-C-aminomethylene-bridged nucleic acid (BNANC) have been directly synthesized from pyrimidine BNANC using transglycosylation reaction with high stereoselectivity and excellent yield. The BNANC purine nucleosides (adenosine, guanosine, and inosine) prepared by the direct and practical procedure were derivatized to the corresponding phosphoramidites and were incorporated into DNA. A thermal denaturation study showed that BNANC-containing oligonucleotides hybridized to RNA selectively with excellent mismatch discrimination.

Despite considerable efforts to develop efficient carriers, the major target organ of short-interfering RNAs (siRNAs) remains limited to the liver. Expanding the application outside the liver is required to increase the value of siRNAs. Here we report on a novel platform targeted to muscular organs by conjugation of siRNAs with anti-CD71 Fab' fragment. This conjugate showed durable gene-silencing in the heart and skeletal muscle for one month after intravenous administration in normal mice. In particular, 1μg siRNA conjugate showed significant gene-silencing in the gastrocnemius when injected intramuscularly. In a mouse model of peripheral artery disease, the treatment with myostatin-targeting siRNA conjugate by intramuscular injection resulted in significant silencing of myostatin and hypertrophy of the gastrocnemius, which was translated into the recovery of running performance. These data demonstrate the utility of antibody conjugation for siRNA delivery and the therapeutic potential for muscular diseases.

We previously demonstrated that dibenzoylhydrazines (DBHs) are not only P-glycoprotein (P-gp) substrates, but also inhibitors. In the present study, we evaluated the inhibition of P-gp-mediated quinidine transport by two series of DBHs and performed a classical QSAR analysis and docking simulation in order to investigate the mechanisms underlying P-gp substrate/inhibitor recognition. The results of the QSAR analysis identified the hydrophobic factor as the most important for inhibitory activities, while electronic and steric effects also influenced the activities. The different substituent effects observed in each series suggested the different binding modes of each series of DBHs, which was supported by the results of the docking simulation.

Almost all highly metastatic tumor cells possess high platelet aggregating abilities, thereby form large tumor cell-platelet aggregates in the microvasculature. Embolization of tumor cells in the microvasculature is considered to be the first step in metastasis to distant organs. We previously identified the platelet aggregation-inducing factor expressed on the surfaces of highly metastatic tumor cells and named as Aggrus. Aggrus was observed to be identical to the marker protein podoplanin (alternative names, T1α, OTS-8, and others). Aggrus is frequently overexpressed in several types of tumors and enhances platelet aggregation by interacting with the platelet receptor C-type lectin-like receptor 2 (CLEC-2). Here, we generated a novel single-chain antibody variable region fragment (scFv) by linking the variable regions of heavy and light chains of the neutralizing anti-human Aggrus monoclonal antibody MS-1 with a flexible peptide linker. Unfortunately, the generated KM10 scFv failed to suppress Aggrus-induced platelet aggregation in vitro. Therefore, we performed phage display screening and finally obtained a high-affinity scFv, K-11. K-11 scFv was able to suppress Aggrus-induced platelet aggregation in vitro. Moreover, K-11 scFv prevented the formation of pulmonary metastasis in vivo. These results suggest that K-11 scFv may be useful as metastasis inhibitory scFv and is expected to aid in the development of preclinical and clinical examinations of Aggrus-targeted cancer therapies.

P-glycoprotein (P-gp) is a member of the ATP-binding cassette transporter family. It actively transports a wide variety of compounds out of cells to protect humans from xenobiotics. Thus, determining whether chemicals are substrates and/or inhibitors of P-gp is important in risk assessments of pharmacokinetic interactions among chemicals because P-gp-mediated transport processes play a significant role in their absorption and disposition. We previously reported that dibenzoylhydrazines (DBHs) such as tebufenozide and methoxyfenozide (agrochemicals) stimulated P-gp ATPase activity. However, it currently remains unclear whether these derivatives are transport substrates of P-gp and inhibit transport of other chemicals by P-gp. In the present study, in order to evaluate the interactions of DBHs with other chemicals in humans, we determined whether DBHs are P-gp transport substrates using both the in vitro bidirectional transport assay and the in vivo study of rats. In the in vivo study, we investigated the influence of P-gp inhibitors on the brain to plasma ratio of methoxyfenozide in rats. We also examined the inhibitory effects of DBHs on quinidine (a P-gp substrate) transport by P-gp in order to ascertain whether these derivatives are inhibitors of P-gp. Based on the results, DBHs were concluded to be weak P-gp transport substrates and moderate P-gp inhibitors. However, the risk of DBHs caused by interaction with other chemicals including drugs was considered to be low by considering the DBHs' potential as the substrates and inhibitors of P-gp as well as their plasma concentrations as long as DBHs are properly used.

Objectives: MicroRNAs (miRNAs) are small functional RNAs that regulate mRNAs for degradation or translational suppression. In the present study, we aimed to reveal functional importance of miRNA-494 (miR-494) in A549 human lung cancer cells. Materials and methods: We established A549 cells that constitutively expressed miR-494. Next, we sought to investigate insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) mRNA as an miR-494 target. For this, we constructed a reporter plasmid bearing potential miR-494 binding sequences derived from the 3′-untranslated region (3′-UTR) of IGF2BP1 mRNA in the 3′-UTR of the luciferase gene. Results: Through comparison between miR-494 expressing cells and control cells, we revealed that miR-494 suppressed cell proliferation and colony forming activity, and induced senescence. Reporter activity was inhibited by miR-494. In addition, IGF2BP1 mRNA levels were down-regulated in A549 cells that constitutively expressed miR-494. IGF2BP1 has been shown to bind and suppress IGF2 mRNA, and this could be a reason why IGF2BP1 can regulate cell function. Therefore, we analysed IGF2 mRNA levels and revealed that IGF2 was up-regulated in A549 cells that constitutively expressed miR-494. Finally, elevated IGF2 mRNA levels in A549 cells that constitutively expressed miR-494 were suppressed to basal level by an miR-494 inhibitor. Conclusions: Taken together, IGF2BP1 and its downstream target IGF2 could be a crucial axis for miR-494 in regulation of the destiny of A549 cells.

The transcription factors DP1 and DP2 have been implicated in crucial gene regulation as heterodimer partners of E2F; however, the functional differences between DP1 and DP2 remain poorly understood. To gain insight into DPs in human somatic cells, we first suppressed endogenous DP1 and DP2 using RNA interference and examined the effect of their loss on gene expression changes in HeLa cervical cancer cells. A DNA microarray and gene pathway analysis revealed that the suppression of well-known E2F/DP-regulated pathways, including the G1 to S phase transition of the cell cycle and DNA replication, was manifested in accordance with the acute loss of DP1 and DP2. On the other hand, the acute loss of DP1 and DP2 increased the p21Waf1/Cip1 mRNA level compared with the control RNA treatment. We further showed that the inactivation of DP1, but not DP2, resulted in mRNA induction for p53, an upstream regulator of p21Waf1/Cip1. Furthermore, in A549 lung cancer cells as well as HeLa cells, the mRNA and protein levels of p53 and p21Waf1/Cip1 were stabilized specifically upon DP1 depletion, whereas p53-regulated apoptotic factor BAX mRNA was unchanged. Finally, the impairment of DP1, but not DP2, increased senescence in HeLa, A549 and WI-38 diploid fibroblasts but not in p53 null Saos-2 osteosarcoma cells. Taken together, these results suggest that DP1, but not DP2, is uniquely involved in the regulation of the p53 and p21Waf1/Cip1 pathway, thereby augmenting senescence in human somatic cells. Copyright © 2011 John Wiley & Sons, Ltd.

For more effective DNA triplex formation under neutral conditions, we synthesized triplex-forming oligonucleotides containing 8-thioxodeoxyadenosine (s(8)dA) residues in place of the protonated deoxycytidines required for the third base pairing with DNA duplexes. Consequently, it was found that s(8)dA exhibited much stronger hybridization ability than dC under neutral conditions when four s(8)dA bases were arranged in a consecutive sequence.

We propose a new strategy called the 'Protected DNA Probes (PDP) method' in which appropriately protected bases selectively bind to the complementary bases without the removal of their base protecting groups. Previously, we reported that 4-N-acetylcytosine oligonucleotides (ac(4)C) exhibited a higher hybridization affinity for ssDNA than the unmodified oligonucleotides. For the PDP strategy, we created a modified adenine base and synthesized an N-acylated deoxyadenosine mimic having 6-N-acetyl-8-aza-7-deazaadenine (ac(6)az(8)c(7)A). It was found that PDP containing ac(4)C and ac(6)az(8)c(7)A exhibited higher affinity for the complementary ssDNA than the corresponding unmodified DNA probes and showed similar base recognition ability. Moreover, it should be noted that this PDP strategy could guarantee highly efficient synthesis of DNA probes on controlled pore glass (CPG) with high purity and thereby could eliminate the time-consuming procedures for isolating DNA probes. This strategy could also avoid undesired base-mediated elimination of DNA probes from CPG under basic conditions such as concentrated ammonia solution prescribed for removal of base protecting groups in the previous standard approach. Here, several successful applications of this strategy to single nucleotide polymorphism detection are also described in detail using PDPs immobilized on glass plates and those prepared on CPG plates, suggesting its potential usefulness.

New bi- and tricyclic deoxycytidine derivatives (dChpd, dCmpp, dCtpp, dCppp) were synthesized as analogues of a fluorescent nucleoside, dChpp, previously reported. The carbamoyl group of dChpd and the 5-position of the cytosine ring are bridged via an ethylene linker so that the modified group forms a nonplanar structure with the cytosine ring. The fluorescent study of dChpd indicated that the coplanar structure between the carbamoyl group and the cytosine ring is of importance. N-Methylation of the carbamoyl group (dCmpp) weakened the intensity of the fluorescence of dChpp, and the derivative (dCtpp), which had a thiocarbamoyl group, lost its fluorescent property. Moreover, addition of a pyrrolo-ring (dCppp) to dChpp enhanced the intensity of fluorescence, and an emission light was observed with a marked Stokes shift of 120 nm.

Deoxycytidine derivatives modified with various N-substituted carbamoyl groups at the 4-amino group were synthesized. The detailed 1H NMR studies suggest that the 3′,5′-O-disilylated N-carbamoyldeoxycytidine derivative 11 exists as a species having an intramolecular hydrogen bond between the N3 atom and the carbonyl oxygen atom in MeOD but upon addition of CDCl3, the amount of a homodimer species, having intermolecular hydrogen bonds, gradually increases. Oligodeoxyribonucleotides incorporating various 4-N-carbamoyldeoxycytidine derivatives were also synthesized. These modified oligodeoxynucleotides can hybridize with the complementary strands without disturbing the structure of DNA duplexes, hence changing the orientation of the carbamoyl group in such a manner that a stable Watson–Crick base pair can be formed with the guanine base at the opposite site. It should be noted that the base recognition ability (G against T, C and A) of these modified cytosine bases can be preserved satisfactorily. These results suggest that the 4-N-carbamoyl group is useful as a backbone structure of the linker between various functional residues and oligonucleotides. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2006)

[reaction: see text] A new route to N-unprotected deoxynucleoside 3'-phosphoramidite building blocks by use of highly selective N-deacylation of commercially available N-acylated deoxynucleoside 3'-phosphoramidites is described. These compounds could be readily converted to other types of N-protected species by facile N-acylations with acylating reagents.

Modified oligodeoxynucleotides incorporating 4- N -( N -arylcarbamoyl)-dC derivatives 1a – c were synthesized. The 1 H NMR spectra of 1a – c suggest that the carbamoyl group forms an intramolecular hydrogen bond with the cytosine ring nitrogen atom so that formation of a Watson–Crick base pair with the complementary guanine base is inhibited. The hybridization properties of oligodeoxynucleotides containing 1a – c were investigated by use of T m analysis. The hybridization properties of 4- N -( N -phenylcarbamoyl)-dC ( 1a ) were similar to those of 4- N -( N -alkylcarbamoyl)-dC derivatives reported previously. In sharp contrast to 1a , it turned out that 4- N -( N -napht-1-yl) and ( N -quionol-5-yl)-dC ( 1b , c ) have a unique property as a universal base.

Oligodeoxyribonucleotides incorporating 4-N-alkoxycarbonyldeoxycytidine derivatives were synthesized on polystyrene-type ArgoPore resins having a new benzyloxy(diisopropyl)silyl linker, by use of ZnBr(2) as the detritylating agent. The first 3'-terminal thymidine could be attached to the resin by successive in situ reactions of 5'-O-DMTr-thymidine with diisopropylsilanediyl ditriflate and an ArgoPore resin containing hydroxyl groups. The use of this new silanediyl-type linker allowed release of the DNA chain from the resin by treatment with TBAF under neutral conditions. The T(m) experiments apparently showed that incorporation of 4-N-alkoxycarbonyldeoxycytidines into DNA strands resulted in higher hybridization affinity with the complementary DNA strands than that of 4-N-acyldeoxycytidines. In addition, comparable T(m) studies using oligodeoxyribonucleotides incorporating acyl (RC(O)-) groups and alkoxyacyl (RO(CH(2))(n)C(O)-) groups having the same chain length show that the latter tend to exhibit higher T(m) values than the former. It turned out that 4-N-alkoxycarbonyldeoxycytidines can form base pairs not only with deoxyguanosine but also with deoxyadenosine. Based on the ab initio calculations of the hydrogen bond energies of the possible base pairs formed between 4-N-methoxycarbonyl-1-methylcytosine and 9-methyladenine and the NMR analysis of the base-pairs of (15)N-labeled 4-N-alkoxycarbonyldeoxycytidines with deoxyadenosine derivatives, we conclude that the base pair involves two unique hydrogen bonds between the cytosyl 4-NH group and the adenyl N(1) atom and between the O atom of the ester group and the adenyl 6-NH group.

A new oxidation method for DNA synthesis was developed by the use of 1,1-dihydroperoxycyclododecane in CH 2 Cl 2 –EtOAc under anhydrous conditions. This new oxidizer was successfully applied to the synthesis of oligodeoxyribonucleotides that involves an oxidation step for conversion of phosphite intermediates to phosphate derivatives.

Tolvaptan is a competitive vasopressin V2-receptor antagonist that inhibits water reabsorption in the renal collecting ducts. A selective and sensitive liquid chromatography–tandem mass spectrometry method for determining tolvaptan and its nine metabolites in rat serum was developed and validated. An analogue of tolvaptan was used as an internal standard. Sample preparation involved protein precipitation following solid-phase extraction. Chromatographic separation was performed on a C18 reversed-phase column with a linear gradient elution. The flow rate was 0.25 mL/min, and total run time was 30 min. The analytes were detected by tandem mass spectrometry using an electrospray ionization interface in positive ion mode and multiple reaction monitoring. The calibration curve showed linearity over the concentration range from 5 to 1,000 ng/mL for each analyte. The lower limit of quantification using 0.1 mL of rat serum was 5 ng/mL for each analyte. Precision did not exceed 5.7 %, and accuracy as relative error were within ± 7.5 % for all analytes. The validated method was successfully applied to evaluate the pharmacokinetics of oral tolvaptan in rats, indicating the systemic exposure to tolvaptan in females eight times larger than that in males.

The transcription factors DP1 and DP2 have been implicated in crucial gene regulation as heterodimer partners of E2F; however, the functional differences between DP1 and DP2 remain poorly understood. To gain insight into DPs in human somatic cells, we first suppressed endogenous DP1 and DP2 using RNA interference and examined the effect of their loss on gene expression changes in HeLa cervical cancer cells. A DNA microarray and gene pathway analysis revealed that the suppression of well-known E2F/DP-regulated pathways, including the G1 to S phase transition of the cell cycle and DNA replication, was manifested in accordance with the acute loss of DP1 and DP2. On the other hand, the acute loss of DP1 and DP2 increased the p21Waf1/Cip1 mRNA level compared with the control RNA treatment. We further showed that the inactivation of DP1, but not DP2, resulted in mRNA induction for p53, an upstream regulator of p21Waf1/Cip1. Furthermore, in A549 lung cancer cells as well as HeLa cells, the mRNA and protein levels of p53 and p21Waf1/Cip1 were stabilized specifically upon DP1 depletion, whereas p53-regulated apoptotic factor BAX mRNA was unchanged. Finally, the impairment of DP1, but not DP2, increased senescence in HeLa, A549 and WI-38 diploid fibroblasts but not in p53 null Saos-2 osteosarcoma cells. Taken together, these results suggest that DP1, but not DP2, is uniquely involved in the regulation of the p53 and p21Waf1/Cip1 pathway, thereby augmenting senescence in human somatic cells.

A new silyl-type linker for the automated solid-phase synthesis of oligodeoxynucleotides was developed. A hydrosilane-type reagent for introduction of the silyl linker was synthesized in an overall yield of 50% from 1,4-dibromobenzene. By using this linker, an oligonucleotide having base-labile protecting groups could be isolated from the controlled pore glass under neutral conditions.

For more effective DNA triplex formation under neutral conditions, we synthesized triplex-forming oligonucleotides (TFO) containing 8-thioxodeoxyadenine (s(8)A) residues in place of the protonated cytosines (Cs) required for the third base pairing with DNA duplexes. Consequently, it was found that s(8)A exhibited much stronger hybridization ability than C under neutral conditions when four s(8)A bases were arranged in a consecutive sequence. Moreover, we also synthesized TFOs containing 5-methyl-2-thiocytosines and examined their ability of triplex formation.

It was found that N-arylcarbamoyl and N-(phenylsulfonyl)carbamoyl (psc) groups could be effectively introduced onto the amino groups of deoxycytidine and deoxyadenosine derivatives and could be removed thermolytically. We succeeded in synthesizing DNA probes incorporating these thermo-removable protecting groups and developed a new system for molecular switching by changing the protection- and deprotection-modes using simple heating and re-carbamoylation with isocyanates. This reversible process enabled us to control the hybridization ability of the DNA probes.

New 5-modified pyrimidine derivatives that were expected to form Hoogsteen-type triplexes have been synthesized, 5-pyrrolyl-2'-O-methylcytosine was designed as a protonated cytosine mimic to realize pH-independent triplex formation with the complementary G-C Watson-Crick base pair by inducing the imino tautomer of cytosine. 5-Pyrrolyl-2'-O-methyluridine was also synthesized to enhance the stacking effect for stabilization of triplexes. For the deprotection of the DMTr group in the phosphoramidite method, EtSi3H was added as the scavenger of the dimethoxytrityl-cation to avoid side-reactions on the pyrrolyl group. The triplex forming ability of TFOs containing modified nucleosides was evaluated by Tm experiments under various conditions.

A new fluorescent deoxycytidine analog, 10-(2-deoxy-beta -D-ribofuranosyl)-pyrimido[4',5' :4,5]-pyrimido[1,6-a]indole-6,9(7H)-dione (dC(PPI)) was synthesized. Its fluorescent properties were studied in detail. It was found that this fluorescent nucleoside dC(PPI) could be used as a fluorescent label for DNA probes with minimal disturbance of their overall structure.

Previously, we observed thermal elimination of a quinolylcarbamoyl group from N-quinolylcarbamoylated nucleobase residues. In this paper, we examined the thermal stability of several carbamoyl groups on nucleobases and developed new carbamoyl-type protecting groups for base protection that can be removed by "heating". Particularly, the phenylsulfonylcarbamoyl group was found to be rapidly removed from three kinds of nucleobases, cytosine, adenine, and guanine bases. Moreover, we could carry out the synthesis of oligodeoxynucleotides by using the thermolytic protecting group for base protection.

10-(2-Deoxy-beta-D-ribofuranosyl)pyrimido[4',5':4,5]pyrimido[1,6-a]indole-6,9(7H)-dione (dCPPI) and its derivatives were synthesized via the Suzuki-Miyaura coupling reaction of 5-iododeoxycytidine with 5-substituted N-Boc-indole-2-borates and characterized by UV-vis and fluorescence spectroscopy. The new fluorescent nucleosides showed rather large Stokes shifts (116-139 nm) in an aqueous buffer. The fluorescent intensities were dependent on the nature of the substituents on the indole rings. The electron-withdrawing groups increased the fluorescent intensity while the electron-donating groups having lone pairs decreased it. Among the substituted dCPPI derivatives tested, the trimethylammonium derivative of dCPPI was found to emit the brightest fluorescent light. The solvatochromism of dCPPI and its derivatives was also studied. Some of the dCPPI derivatives showed interesting solvent-dependent fluorescence enhancement and could be useful as new fluorescent structural probes for nucleic acids. The Lippert-Mataga analyses of the Stokes shift were also carried out to obtain estimated values of the dipole moment of the excited states of some of the derivatives.