Influence of 3'-azido-2',3'-dideoxyguanosine treatment on telomere length in human telomerase-immortalized human fibroblast cells.

In order to study telomerase activation in normal cells, a telomerase-immortalized fibroblast cell line, hTERT-BJ1, treated with a telomerase inhibitor, 3'-azido-2',3'-dideoxyguanosine (AZddG), is considered to be a good model. Long-term treatment with AZddG resulted in telomere shortening from 10-20 kbp to 5-6 kbp in cultured hTERT BJ1 cells. However, the telomere length then stabilized. As expected, removal of AZddG from the culture medium induced telomere lengthening in the cells, suggesting that telomerase activity was recovered upon AZddG removal. The effect of recovery of telomerase activity in hTERT-BJ1 cells will be investigated in future studies.

Influence of 3'-azido-2',3'-dideoxyguanosine treatment on amounts of TERT and POT1 in human HL60 cells.

In human cells, TERT (telomerase reverse transcriptase) is involved in the synthesis of telomere DNA, and POT1 (protection of telomeres 1) is believed to be a regulator of telomere length. We have reported that long-term treatment of human HL60 cells with 50 microM 3'-azido-2',3'-dideoxyguanosine (AZddG) caused telomeres to shorten significantly during early passages (up to 40-50 days), but that telomere length was then stabilized at approximately 2 kbp during later passages. Additionally, cell growth rates showed no obvious change during culture in the presence of 50 microM AZddG. Western blot analysis of these cells showed that the amounts of TERT and POT1 expressed were increased significantly and slightly, respectively. Furthermore, telomeric 3' G-overhangs (G-tails) of AZddG-treated cells were lengthened. These findings suggest that HL60 cells may develop resistance to telomere erosion induced by AZddG.

Telomere shortening in human HL60 cells by treatment with 3'-azido-2',3'-dideoxynucleosides and telomerase inhibition by their 5'-triphosphates.

Telomerase is thought to play an important role in the mechanism of tumor cell immortalization by maintenance of telomere length. To obtain information on the susceptibility of telomerase to nucleoside analogues, the effects of base-modified 3'-azido-2',3'-dideoxynucleoside triphosphates on the enzyme were investigated. It is suggested that the 2-amino group of the nucleotide purine nucleus is important for the inhibitory activity. Telomere shortening caused by long-term treatment with these nucleosides is also described.

Influence of nucleoside analogue treatment on telomere length and TRF2 amount in human HL60 cells.

Long-term treatment with 3'-azido-2',3'-dideoxy-guanosine (AZddG) results in reproducible telomere shortening in cultured human HL60 cells. TRF2 protein has been implicated in the protection of chromosome ends. It binds to double-strand repeats and may have an indirect role in protecting the G-rich overhang by recruiting other telomere-binding proteins to the G-tail or by mediating the formation of the telomeric t-loop. Western blot analysis demonstrated no change or a slight increase, of the TRF2 protein level in HL60 cells with AZddG-induced telomere shortening. The effects of nucleoside analogues on TRF2 suggest that it is not telomere length per se, but rather the presence or absence of a protective telomere state, which determines whether senescence ensues.

3'-Azido-2',3'-dideoxynucleoside 5'-triphosphates inhibit telomerase activity in vitro, and the corresponding nucleosides cause telomere shortening in human HL60 cells.

Telomerase adds telomeric DNA repeats to the ends of linear chromosomal DNA. 3'-Azido-3'-deoxythymidine 5'-triphosphate (AZTTP) is a known telomerase inhibitor. To obtain more selective and potent inhibitors that can be employed as tools for studying telomerase, we investigated the telomerase-inhibitory effects of purine nucleosides bearing a 3'-down azido group: 3'-azido-2',3'-dideoxyguanosine (AZddG) 5'-triphosphate (AZddGTP), 3'-azido-2',3'-dideoxy-6-thioguanosine (AZddSG) 5'-triphosphate (AZddSGTP), 3'-azido-2',3'-dideoxyadenosine (AZddA) 5'-triphosphate (AZddATP) and 3'-azido-2',3'-dideoxy-2-aminoadenosine (AZddAA) 5'-triphosphate (AZddAATP). Of these, AZddGTP showed the most potent inhibitory activity against HeLa cell telomerase. AZddGTP was significantly incorporated into the 3'-terminus of DNA by partially purified telomerase. However, AZddGTP did not exhibit significant inhibitory activity against DNA polymerases alpha and delta, suggesting that AZddGTP is a selective inhibitor of telomerase. We also investigated whether long-term treatment with these nucleosides could alter telomere length and growth rates of human HL60 cells in culture. Southern hybridization analysis of genomic DNA prepared from cells cultured in the presence of AZddG and AZddAA revealed reproducible telomere shortening.

Telomerase inhibition by 3'-azido-2', 3'-dideoxynucleoside 5'-triphosphates and telomere shortening in human cultured cells by the corresponding nucleosides.

Telomerase is believed to be a good target for the development of antitumor agents. In this study, 3'-azido-2',3'-dideoxy-2-aminoadenosine (AZddAA), 3'-azido-2',3'-dideoxyadenosine (AZddA), 9-(3-azido-2,3-dideoxy-beta-D-ribofuranosyl)-2-aminopurine (AZddAP), 3'-azido-2-chloro-2',3'-dideoxyadenosine (AZddClA) and their triphosphate derivatives were synthesized. Telomerase assay studies showed that the 2-amino group plays an important role in the inhibitory activity of these compounds. In addition, AZddAA was found to cause telomere shortening in of HL60 cells in culture.

Synthetic nucleosides and nucleotides. 43. Inhibition of vertebrate telomerases by carbocyclic oxetanocin g (C.OXT-G) triphosphate analogues and influence of C.OXT-G treatment on telomere length in human HL60 cells.

Telomerase, responsible for telomere synthesis, is expressed in approximately 90% of human tumor cells but seldom in normal somatic cells. In this study, inhibition by carbocyclic oxetanocin G triphosphate (C. OXT-GTP) and its analogues was investigated in order to clarify the susceptibility of telomerase to various nucleotide analogues. C. OXT-GTP competitively inhibited telomerase activity with respect to dGTP However, C. OXT-GTP had a potent inhibitory effect on DNA polymerase alpha. It was examined whether the nucleoside (C. OXT-G) was able to alter telomere length in cultured human HL60 cells. Contrary to expectation, long-term treatment with 10 microM C. OXT-G was found to cause telomere lengthening.

Influence of long-term treatment with cytosine arabinoside on telomere length in human HL60 cells.

Immortal tumor cells employ a telomere length maintenance mechanism to escape the normal limits on proliferation. We investigated whether treatment with cytosine arabinoside (AraC), whose triphosphate derivative AraCTP might partially inhibit the synthesis of C-rich telomere strands, is effective for inducing telomere shortening in human HL60 cells. Long-term treatment with 0.01 microM AraC was found to cause significant telomere lengthening, but had no marked effects on cell population doubling rates or morphology.

Telomere shortening in human HL60 cells by treatment with deoxyguanosine analogs.

Telomerase is a cellular endogenous reverse transcriptase thought to play an important role in the maintenance of telomere length. We investigated the effects of 3'-azido-2',3'-dideoxyguanosine (AZddG) and carbocyclic oxetanocin G (C.OXT-G), of which the triphosphate derivatives AZddGTP and C.OXT-GTP show potent telomerase inhibition, on telomere length of human HL60 cells in culture. Although AZddG caused more significant telomere shortening than C.OXT-G, only a slight decrease of cell growth rate was observed.

Inhibition of vertebrate telomerases by the triphosphate derivatives of some biologically active nucleosides.

In order to clarify the effect of the base moiety of nucleotide analogs on telomerase inhibition, triphosphate derivatives of biologically active nucleosides, 3'-azido-3'-deoxythymidine (AZT), 2'-deoxy-2'-fluoroarafuranosylthymine (FaraT), acycloguanosine (ACG) and their guanine or thymine counterparts (AZdG, FaraG and ACT, respectively) were investigated. In all of the present cases, guanine derivatives showed more potent inhibition than their thymine counterparts.

Inhibition of vertebrate telomerases by the triphosphate derivatives of carbocyclic oxetanocin analogs.

Telomerase is a cellular endogenous reverse transcriptase that uses its internal RNA as a template for extension of the telomere repeat, thus maintaining telomere length. In order to clarify the susceptibility of telomerase to triphosphate derivatives of carbocyclic oxetanocins, inhibition by 9-[trans-trans-2,3-bis(hydroxymethyl)cyclobutyl]guanine triphosphate (C.OXT-GTP) and its methylene analog, 9-(cis-3-hydroxymethyl-2-methylenecyclobutyl)guanine triphosphate (m-C.OXT-GTP) was investigated. Both compounds showed potent inhibitory activity. Lineweaver-Burk plot analyses showed that the inhibition mode of these compounds was competitive with dGTP, the Ki values for C.OXT-GTP and m-C.OXT-GTP being 2.0 microM and 4.9 microM, respectively, and thus smaller than the Km of dGTP (11 microM).

Molecular action mode of Hippospongic acid A, an inhibitor of gastrulation of starfish embryos.

Hippospongic acid A (HA-A) is a novel natural triterpene metabolite that exhibits inhibitory activity against the gastrulation of starfish embryos isolated from a marine sponge, Hippospongia sp. We succeeded in chemically synthesizing the natural enantiomer and the racemate HA-A. In this study, we examined its action mode in vitro. HA-A was a rare compound that could selectively but uniformly inhibit the activities of all the vertebrate DNA polymerases tested such as alpha, beta, delta, epsilon, eta, kappa, and lambda, in the IC(50) range of 5.9-17.6 microM, and interestingly also those of human DNA topoisomerases I and II (IC(50) = 15-25 microM). HA-A exhibited no inhibitory effect on DNA polymerases from insects, plants and prokaryotes, or on many other DNA metabolic enzymes. HA-A was an inhibitor specific to DNA polymerases and DNA topoisomerases from vertebrates, but not selective as to a subclass species among the enzymes. Since DNA polymerase beta is the smallest, we used it to analyze the biochemical relationship with HA-A. Biochemical, BIAcore and computer modeling analyses demonstrated that HA-A bound selectively to the N-terminal 8 kDa DNA template-binding domain of DNA polymerase beta, and HA-A inhibited the ssDNA binding activity. HA-A could prevent the growth of NUGC-3 cancer cells at both the G1 and G2/M phases, and induce apoptosis in the cells. The LD(50) value was 9.5 microM, i.e. in the same range as for the enzyme inhibition. Therefore, we concluded that one molecular basis of the gastrulation of starfish embryos is a process that requires DNA polymerases and DNA topoisomerases, and subsequently the gastrulation was inhibited by HA-A. We also discussed the in vivo role of HA-A.

Kohamaic acid A, a novel sesterterpenic acid, inhibits activities of DNA polymerases from deuterostomes.

We previously found and isolated a novel natural product, designated kohamaic acid A (KA-A), which inhibited the first cleavage of fertilized sea urchin eggs. In this paper, we report that this compound could selectively inhibit the activities of DNA polymerases (pol. alpha, beta, gamma, delta and epsilon ) only from species in the deuterostome branch in the animal kingdom, like sea urchin, fish and mammals, but not from protostomes including insects (fruit fly, Drosophila melanogaster) and mollusks (octopus and oyster). Inhibition of deuterostome DNA polymerases was dose dependent. IC(50) values for DNA polymerases of mammals and fish occurred at approximately 5.8-14.9 microM and those of sea urchin at 6.1-30.3 microM. In the sea urchin DNA polymerases, the activities of the replicative DNA polymerases such as alpha, delta and epsilon were more strongly inhibited than that of the repair-related pol. beta. KA-A is an inhibitor of replicative DNA polymerases from the deuterostome species, and subsequently, the inhibition of the first cleavage of fertilized sea urchin eggs might occur as a result of the suppression of DNA replication.

A sulphoquinovosyl diacylglycerol is a DNA polymerase epsilon inhibitor.

Sulphoquinovosyl diacylglycerol (SQDG) was reported as a selective inhibitor of eukaryotic DNA polymerases alpha and beta [Hanashima, Mizushina, Ohta, Yamazaki, Sugawara and Sakaguchi (2000) Jpn. J. Cancer Res. 91, 1073-1083] and an immunosuppressive agent [Matsumoto, Sahara, Fujita, Shimozawa, Takenouchi, Torigoe, Hanashima, Yamazaki, Takahashi, Sugawara et al. (2002) Transplantation 74, 261-267]. The purpose of this paper is to elucidate the biochemical properties of the inhibition more precisely. As expected, SQDG could inhibit the activities of mammalian DNA polymerases such as alpha, delta, eta and kappa in vitro in the range of 2-5 micro M, and beta and lambda in vitro in the range of 20-45 micro M. However, SQDG could inhibit only mammalian DNA polymerases epsilon (pol epsilon) activity at less than 0.04 micro M. SQDG bound more tightly to mammalian pol epsilon than the other mammalian polymerases tested. Moreover, SQDG could inhibit the activities of all the polymerases from animals such as fish and insect, but not of the polymerases from plant and prokaryotes. SQDG should, therefore, be called a mammalian pol epsilon-specific inhibitor or animal polymerase-specific inhibitor. To our knowledge, this represents the first report about an inhibitor specific to mammalian pol epsilon.

Distribution and molecular evolution of rhamnose-binding lectins in Salmonidae: isolation and characterization of two lectins from white-spotted Charr (Salvelinus leucomaenis) eggs.

L-Rhamnose-binding lectins were isolated from white-spotted charr (Salvelinus leucomaenis) eggs to understand the distribution and molecular evolution of the lectins in Salmonidae. Only two L-rhamnose-binding lectins, named WCL1 and WCL3, were isolated from white-spotted charr eggs, though three lectins, named STL1, STL2, and STL3, had been obtained from steelhead trout (Oncorhynchus mykiss) eggs. The cDNAs of WCL1 and WCL3 included 1,245 and 838 bp nucleotides with open reading frames of 933 and 651 nucleotides, respectively, and encoded for the complete amino acid sequences of mature proteins consisted of 288 (WCL1) and 195 (WCL3) residues, and signal sequences of 23 and 22 residues, respectively. WCLs were composed of three (for WCL1) or two (for WCL3) tandemly repeated homologous domains, which consisted of about 95 amino acid residues, and showed 91 and 93% sequence identities to STL1 and STL3, respectively. The mRNAs of WCL1 and WCL3 were detected exclusively in liver and ovary, respectively, however, neither a protein nor mRNA corresponding to STL2 could be identified in white-spotted charr. The phylogenetic tree of the sequences encoding carbohydrate recognition domains of 7 lectins from 4 species shows 5 functional clusters and their evolutional process. These results indicate that multiple L-rhamnose-binding isolectins have diverged by gene duplication and exon shuffling to play various biological roles in each species.

Tissue-specific expression of rhamnose-binding lectins in the steelhead trout (Oncorhynchus mykiss).

Tissue-specific expression of three L-rhamnose-binding lectins, named STL1, STL2, and STL3, in the steelhead trout (Oncorhynchus mykiss) was investigated. STL2 and STL3 mRNAs were restricted in the oocytes. In contrast, STL1 mRNA was detected only in the liver. The transcription of STL2 and STL3 started in previtellogenic oocytes. These results showed distinct expression profiles of rhamnose-binding lectins in the fish.

Rhamnose-binding lectins from steelhead trout (Oncorhynchus mykiss) eggs recognize bacterial lipopolysaccharides and lipoteichoic acid.

The interaction between bacteria and three L-rham-nose-binding lectins, named STL1, STL2, and STL3, from steelhead trout (Oncorhynchus mykiss) eggs was investigated. Although STLs bound to most Gram-negative and Gram-positive bacteria, they agglutinated only Escherichia coli K-12 and Bacillus subtilis among the bacteria tested. The binding was inhibited by L-rhamnose. STLs bound to distinct serotypes of lipopolysaccharides (LPSs), and showed much higher binding activities to smooth-type LPSs of Escherichia coli K-12 and Shigella flexneri 1A than to their corresponding rough-type LPSs. STLs also bound to lipoteichoic acid (LTA) of Bacillus subtilis. These results indicate that STLs bound to bacteria by recognizing LPSs or LTA on the cell surfaces.

Telomerase-inhibitory effects of sugar-modified nucleotide analogs.

Telomerase is an endogenous reverse transcriptase that uses its internal RNA moiety as a template for the synthesis of telomere repeats, thus maintaining telomere length. To study the susceptibility of telomerase to sugar-modified nucleotide analogs, inhibition by arabinofuranosylguanine 5'-triphosphate (araGTP), 3'-azido-2',3'-dideoxyguanosine 5'-triphosphate (AZdGTP), 2',3'-dideoxy-2'-fluoroarabino-furanosylguanine 5'-triphosphate (FaraGTP), and their thymine counterparts was investigated. Among these compounds, all dGTP analogs showed potent inhibitory activity against human telomerase. Conversely, dTTP analogs showed moderate or weak inhibition. Partially purified telomerase from cherry salmon testis utilized ddGTP and AZdGTP as substrates into the 3'-terminus of DNA.

Change in the levels of adenine-related compounds in starfish ovarian follicle cells following treatment with gonad-stimulating substance.

The resumption of meiosis in starfish oocytes is induced by 1-methyladenine (1-MeA), which is produced by ovarian foilicle cells under the influence of a gonad-stimulating substance (GSS). It has been reported that the 1-MeA produced is newly synthesized via a process of methylation, rather than being pre-stored within follicle cells or a breakdown product of some 1-MeA-containing substance. The present study examined a possible substrate for 1-MeA biosynthesis stored in follicle cells of the starfish Asterina pectinifera. Analyses using high-performance liquid chromatography indicated a large source of ATP among the adenine-related compounds in these follicle cells. When follicle cells were incubated in seawater in the presence of GSS, 1-MeA production was stimulated significantly. GSS also caused a reduction in intracellular levels of ATP. There was no change in the levels of either ADP or AMP. The amount of ATP consumed under the influence of GSS was similar to the amount of 1-MeA produced. Methionine and selenomethionine enhanced both 1-MeA production and ATP consumption by GSS in follicle cells. In contrast, ethionine and selenoethionine, competitive inhibitors of methionine, inhibited these processes. These results suggest that ATP is a possible substrate in the biosynthesis of 1-MeA by starfish ovarian follicle cells.