Fumiaki Uchiumi

Transcriptional suppression of the HIV promoter by natural compounds.

Tannins and lignins are natural compounds contained in plants such as tea leaves. Previously, we demonstrated that tannic acid represses 12-o-tetra-decanoyl phorbol-13-acetate (TPA)-induced human immunodeficiency virus (HIV) promoter activity. Furthermore, we demonstrated that a 30-bp element located just downstream of the NF-kappaB element in the HIV promoter responds negatively to tannic acid. However, the kinds of molecules responsible for this suppressive effect have remained unknown, because tannic acid is a mixture of various galloylglucoses. Here, we examined structure-defined natural compounds for HIV promoter-suppressive effects. We found that ellagitannins suppress TPA-induced HIV promoter activity to the same extent as tannic acid. 3-phenylcoumarins, isoflavone and chalcones have more suppressive effects than ellagitannins. On the other hand, other flavonoids and acetogenins have no suppressive effect. 3-phenylcoumarins and chalcones showed no suppressive effect on the cytomegalovirus (CMV) promoter, suggesting that they act specifically on the HIV promoter. These results suggest that 3-phenylcoumarin or chalcone compounds could be used to develop novel anti-HIV drugs with an action targeted at HIV promoter activity.

Novel inhibitors of poly(ADP-ribose) glycohydrolase

The inhibitory effects on poly(ADP-ribose) glycohydrolase purified from human placenta of three classes of chemically defined tannins; gallotannins, ellagitannins and condensed tannins, were examined in vitro. Oligomeric ellagitannins were found to be most potent inhibitors of poly(ADP-ribose) glycohydrolase, their potencies increasing with increasing number of monomeric residues (dimer < trimer < tetramer). Monomeric ellagitannins and gallotannins were less inhibitory. Condensed tannins, which consist of an epicatechin gallate oligomer without a glucose core, were not appreciably inhibitory. A structure-activity study showed that higher-order conformations of the conjugates with glucose of hexahydroxydiphenoyl and valoneoyl groups, which are unique components of ellagitannins, cooperatively potentiated the inhibitory activity.

Identification and Characterization of a Tannic Acid-responsive Negative Regulatory Element in the Mouse Mammary Tumor Virus Promoter

Tannic acid, which comprises polyphenolic compounds from tea leaves, suppresses the glucocorticoid-induced gene expression of mouse mammary tumor virus (MMTV) integrated into 34I cells. To investigate whether this suppression is due to promoter responsiveness to tannic acid, we performed chloramphenicol acetyltransferase analysis transfecting a MMTV promoter containing a chloramphenicol acetyltransferase expression vector into mouse fibroblast L929 cells. Deletion analysis of the promoter region revealed that a 50-base pair (bp) region located downstream of the TATA element is responsible for the suppressive effect of tannic acid. The tannic acid-sensitive suppressibility was introduced into a thymidine kinase promoter by inserting the 50-bp region into the region on the 5′-upstream side of the promoter. Detailed point mutation analyses revealed that two elements, a 13-bp element and an ACTG motif in the 50-bp region, contribute to tannic acid sensitivity and promoter repressibility, respectively. Interestingly, this repressive ACTG motif is found in the human immunodeficiency virus promoter, the activity of which is also suppressed by tannic acid (Uchiumi, F., Maruta, H., Inoue, J., Yamamoto, T., and Tanuma, S. (1996) Biochem. Biophys. Res. Commun. 220, 411–417). Furthermore, electrophoretic mobility shift analysis revealed that a protein factor(s) in nuclear extracts from L929 cells binds to the 50-bp region in a sequence-specific manner and that the amount of DNA-protein complex is increased by tannic acid treatment. Moreover, the negative regulatory sequence ACTG and the tannic acid-sensitive 13-bp element in this region were shown to be responsible for the formation of the DNA-protein complex by electrophoretic mobility shift analysis and footprint analyses. These findings suggest that the suppressive effect of tannic acid on MMTV gene expression is mediated by a protein factor(s) that binds to the negative regulatory element containing the common ACTG motif in a cooperative manner with the tannic acid-sensitive 13-bp element.

Characterization of 5’-Flanking Regions of Various Human Telomere Maintenance Factor-Encoding Genes

Telomeres are the unique nucleoprotein complex structures located at the end of linear eukaryotic chromosomes (Blackburn, 2000; de Lange, 2006). They are composed of TTAGGG repeats that are typically 10 kb at birth and gradually shorten with cell divisions (de Lange, 2006). Telomerase is composed of the protein subunit TERT and the RNA subunit TERC (TR). It elongates the telomere by adding telomeric repeats (Greider & Blackburn, 1987). The 50 to 300 nucleotides from the terminal end of the telomeres are single stranded 3’-protluded Goverhang structures which make the t-loop configuration (de Lange, 2006; Griffith et al., 1999). Mammalian telomeres are included in heterochoromatin and attached to the nuclear matrix (Oberdoerffer & Sinclair, 2007; Gonzalez-Suarez & Gonzalo, 2008). Telomere shortening causes instability of the ends of chromosomes to lead to replicative senescence (O’Sullivan & Karlseder, 2010; Lundblad & Szostak, 1989). Therefore, the ends of telomeres should be protected from damaging or cellular activities. The t-loop structures are regulated by shelterin protein factors, TRF1, TRF2, Rap1, TIN2, TPP1, POT1 (Gilson & Geli, 2007; O’Sullivan & Karlseder, 2010), and Rec Q DNA helicases, WRN and BLM (Chu & Hickson, 2009). TRF1 and TRF2, which bind to duplex telomeric DNA and retain shelterin on the telomere repeats, were shown to interact with various functional proteins (Giannone et al., 2010). Molecular structural analysis of Rap1 revealed that its mechanism of action involves interaction with TRF2 and Taz1 proteins (Chen et al., 2011). A recent study showed that depletion of TPP1 and its partner TIN2 causes a loss of telomerase recruitment to telomeres (Abreu et al., 2010). POT1 is an important regulator of telomerase length, in stimulating the RecQ helicases WRN and BLM (Opresko et al., 2005). Tankyrase-1 (TANK1), which is classified as a poly(ADP-ribose) polymerase family protein, is also known to regulate telomere homeostasis by modifying TRF1 (Smith et al., 1998; Schreiber et al., 2006). Dyskerin, which is encoded by the DKC1 gene, is a key auxiliary protein that is contained in a Cajal body with TERT (Cohen et al., 2007). Defects in the shelterin components and telomerase are thought to down-regulate telomere structure

Sodium benzylideneascorbate induces apoptosis in HIV‐replicating U1 cells

U1 cells, a subclone of U937 cells chronically infected with human immunodeficiency virus type 1 (HIV‐1), produced HIV‐1 only in the presence of inducers such as 12‐O‐tetradecanoxylphorbol 13‐acetate (TPA) or tumor necrosis factor (TNF)‐α. The expression of HIV‐antigen on U1 cells induced by TPA or TNF‐α was found to be prevented by sodium 5,6‐benzylidene‐l‐ascorbate (SBA) in a concentration‐dependent manner. Treatment of U1 cells with SBA in the presence of inducers resulted in cell death with cell shrinkage, chromatin condensation and DNA fragmentation into nucleosomal oligomers, characteristics of apoptosis. In contrast, SBA had scarcely any apoptotic effect on U1 cells in the absence of inducers. SBA did not also induce apoptosis in parental U937 cells in the presence or absence of inducers. These results suggest that HIV‐replicating U1 cells selectively undergo apoptosis on treatment with SBA.