Studying telomere replication by Q-CO-FISH: the effect of telomestatin, a potent G-quadruplex ligand.

Telomere replication is a critical process for preserving genome integrity. The telomere replication fork proceeds unidirectionally from the last subtelomeric origin towards the end of the chromosome, replicating the 5'-3' G-rich strand by lagging mechanisms and the complementary C-rich strand by leading mechanisms. It has been proposed that the G-rich nature of telomeres may favor the formation of secondary structures such as G-quadruplexes during replication and that specific mechanisms must prevent this to allow the fork to progress unimpeded. The potential of G-quadruplex formation by telomeric sequences has been clearly demonstrated in vitro but it is not known whether these structures form in vivo. We tested the effect of a potent and specific G-quadruplex ligand, telomestatin (TMS), on telomere replication using a novel quantitative approach applied to CO-FISH. We show that TMS, although it penetrates and persists within cells, does not affect telomere replication after short or long-term treatments of mouse embryonic fibroblasts. It does however affect the hybridization efficiency of FISH telomeric probes that recognize the G-rich strand. Our work illustrates the use of a novel technique to measure telomere replication efficiency and suggests that G-quadruplex ligands do not affect telomere replication in a non tumoral context.

Telomerase inhibition with a novel G-quadruplex-interactive agent, telomestatin: in vitro and in vivo studies in acute leukemia.

The telomerase complex is responsible for telomere maintenance and represents a promising neoplasia therapeutic target. Recently, we have demonstrated that treatment with a G-quadruplex-interactive agent, telomestatin reproducibly inhibited telomerase activity in the BCR-ABL-positive leukemic cell lines. In the present study, we investigated the mechanisms of apoptosis induced by telomerase inhibition in acute leukemia. We have found the activation of caspase-3 and poly-(ADP-ribose) polymerase in telomestatin-treated U937 cells (PD20) and dominant-negative DN-hTERT-expressing U937 cells (PD25). Activation of p38 mitogen-activated protein (MAP) kinase and MKK3/6 was also found in telomestatin-treated U937 cells (PD20) and dominant-negative DN-hTERT-expressing U937 cells (PD25); however, activation of JNK and ASK1 was not detected in these cells. To examine the effect of p38 MAP kinase inhibition on growth properties and apoptosis in telomerase-inhibited cells, we cultured DN-hTERT-expressing U937 cells with or without SB203580. Dominant-negative-hTERT-expressing U937 cells stopped proliferation on PD25; however, a significant increase in growth rate was observed in the presence of SB203580. Treatment of SB203580 also reduced the induction of apoptosis in DN-hTERT-expressing U937 cells (PD25). These results suggest that p38 MAP kinase has a critical role for the induction of apoptosis in telomerase-inhibited leukemia cells. Further, we evaluated the effect of telomestatin on the growth of U937 cells in xenograft mouse model. Systemic intraperitoneal administration of telomestatin in U937 xenografts decreased tumor telomerase levels and reduced tumor volumes. Tumor tissue from telomestatin-treated animals exhibited marked apoptosis. None of the mice treated with telomestatin displayed any signs of toxicity. Taken together, these results lay the foundations for a program of drug development to achieve the dual aims of efficacy and selectivity in vivo.

G-Quadruplex stabilization by telomestatin induces TRF2 protein dissociation from telomeres and anaphase bridge formation accompanied by loss of the 3' telomeric overhang in cancer cells.

Inhibition of telomerase activity by telomerase inhibitors induces a gradual loss of telomeres, and this in turn causes cancer cells to enter to a crisis stage. Here, we report the telomerase inhibitor telomestatin, which is known to stabilize G-quadruplex structures at 3' single-stranded telomeric overhangs (G-tails), rapidly dissociates TRF2 from telomeres in cancer cells within a week, when given at a concentration that does not cause normal cells to die. The G-tails were dramatically reduced upon short-term treatment with the drug in cancer cell lines, but not in normal fibroblasts and epithelial cells. In addition, telomestatin also induced anaphase bridge formation in cancer cell lines. These effects of telomestatin were similar to those of dominant negative TRF2, which also causes a prompt loss of the telomeric G-tails and induces an anaphase bridge. These results indicate that telomestatin exerts its anticancer effect not only through inhibiting telomere elongation, but also by rapidly disrupting the capping function at the very ends of telomeres. Unlike conventional telomerase inhibitors that require long-term treatments, the G-quadruplex stabilizer telomestatin induced prompt cell death, and it was selectively effective in cancer cells. This study also identifies the TRF2 protein as a therapeutic target for treating many types of cancer which have the TRF2 protein at caps of the telomere DNA of each chromosome.

Telomerase inhibition, telomere shortening, cell growth suppression and induction of apoptosis by telomestatin in childhood neuroblastoma cells.

Neuroblastoma is a tumour derived from primitive cells of the sympathetic nervous system and is the most common extracranial solid tumour in childhood. Unfavourable tumours are characterised not only by structural changes, including 1p deletion and amplification of the MYCN proto-oncogene, but also by high telomerase activity. Telomeric G-rich single-stranded DNA can adopt in vitro an intramolecular quadruplex structure, which has been shown to inhibit telomerase activity. In this study, we examined telomestatin, a G-quadruplex interactive agent, for its ability to inhibit telomere maintenance of neuroblastoma cells. Telomere length was determined by the terminal restriction fragment method, telomerase activity was measured by a quantitative telomeric repeat amplification protocol, and the expression of human telomerase by quantitative real-time polymerase chain reaction (RT-PCR). Short-term treatment with telomestatin resulted in dose-dependent cytotoxicity and induction of apoptosis. Long-term treatment with telomestatin at non-cytotoxic, but still telomerase activity-inhibiting, concentrations resulted in telomere shortening, growth arrest and induction of apoptosis. These results suggest that the effect of telomestatin is dose-dependent and at least 2-fold. Prolonged low-dose treatment with telomestatin limits the cellular lifespan of NB cells through disruption of telomere maintenance.

A G-quadruplex-interactive agent, telomestatin (SOT-095), induces telomere shortening with apoptosis and enhances chemosensitivity in acute myeloid leukemia.

Telomerase, the ribonucleoprotein enzyme maintaining the telomeres of eukaryotic chromosomes, is up-regulated in the vast majority of human neoplasias but not in normal somatic tissues. Therefore, the telomerase complex represents a promising universal therapeutic target in cancer. Telomeric G-rich single-stranded DNA can adopt in vitro an intramolecular quadruplex structure, which has been shown to inhibit telomerase activity. We examined G-quadruplex interactive agent, telomestatin (SOT-095), for its ability to inhibit the proliferation of human leukemia cells, including freshly obtained leukemia cells. Telomere length was determined by either the terminal restriction fragment method or flow-FISH, and apoptosis was assessed by flow cytometry. Moreover, chemosensitivity was examined in telomestatin-treated U937 cells before ultimate telomere shortening. Treatment with telomestatin reproducibly inhibited telomerase activity in U937 and NB4 cells followed by telomere shortening. Enhanced chemosensitivity toward daunorubicin and cytosine-arabinoside was observed in telomestatin-treated U937 cells, before ultimate telomere shortening. Telomere shortening associated with apoptosis by telomestatin was evident in some freshly obtained leukemia cells from acute myeloid leukemia patients, regardless of sub-types of AML and post-myelodysplasia AML. These results suggest that disruption of telomere maintenance by telomestatin limits the cellular lifespan of AML cells, as well. However, in a minority of AML patients apoptosis was not evident, thus indicating that resistant mechanism might exist in some freshly obtained AML cells. Therefore, further investigation of telomestatin as a therapeutic agent is warranted.

Ammocidin, a new apoptosis inducer in Ras-dependent cells from Saccharothrix sp. I. Production, isolation and biological activity.

A new apoptosis inducer, ammocidin, was isolated from the culture broth of Saccharothrix sp. AJ9571. Ammocidin induced apoptotic cell death in Ras-dependent Ba/F3-V12 cells with an IC50 of 66 ng/ml. No cell death was observed in IL-3-dependent Ba/F3-V12 cells at less than 100 microg/ml of ammocidin. Ammocidin significantly reduced the phosphorylation level of MAPK and S6K that mediate the anti-apoptotic function of Ras.

Ammocidin, a new apoptosis inducer in ras-dependent cells from Saccharothrix sp. II. Physico-chemical properties and structure elucidation.

The structure of ammocidin, a new apoptosis inducer in Ras-dependent cells from Saccharothrix sp. AJ9571, was elucidated to be as shown in Fig. 1 by NMR and degradation studies. Ammocidin consists of a 20-membered macrolide ring and three deoxy sugars identified as 6-deoxy-L-glucose, D-digitoxose and D-olivomycose.

Time courses of increased expression of signaling transduction molecules induced by basic fibroblast growth factor in PC12 cells.

We previously demonstrated that basic fibroblast growth factor (bFGF) protected neuronal injury in in vivo experimental cerebral ischemia. The precise molecular mechanisms of the neuroprotective effect of bFGF, however, remains unsolved. We investigated time courses of up-regulated molecules involved in intracellular signaling transduction pathways induced by bFGF in PC12 cells to explore the possible neuroprotective mechanism of bFGF action. In Western blot analysis, bFGF increased expression of Ras mainly in the early stage up to 24h, returning to the baseline level at 48 h. Expression of phosphatidylinositol 3-kinase (PI 3-kinase) was enhanced throughout the early and later stages, and was more up-regulated at 48 h compared to 24 h. The present findings suggest that bFGF might promote cell survival or proliferation mainly via Ras, and PI 3-kinase might be involved in cell survival and differentiation in PC12 cells.

Neuroprotectins A and B, bicyclohexapeptides protecting chick telencephalic neuronal cells from excitotoxicity. I. Fermentation, isolation, physico-chemical properties and biological activity.

Glutamate, an excitatory amino acid, is known to induce neurotoxicity in central nervous system under abnormal conditions such as ischemia, hypoglycemia, epilepsy, Huntington's chorea, Parkinson's disease and Alzheimer's disease. In our search for neuroprotective agents of microbial origin against excitatory neurotoxins, we have isolated two new bicyclohexapeptides, neuroprotectins A and B, together with a known compound complestatin, from the fermentation broth of Streptomyces sp. Q27107. Neuroprotectins protected primary cultured chick telencephalic neurons from glutamate- and kainate-induced excitotoxicities in a dose-dependant fashion.

Neuroprotectins A and B, bicyclohexapeptides protecting chick telencephalic neurons from excitotoxicity. II. Structure determination.

In the course of our search for neuroprotective agents of microbial origin against kainate-induced neurotoxicity, we have succeeded in the isolation of two new bicyclohexapeptides, neuroprotectins A and B, together with a known compound, complestatin, from the fermentation broth of Streptomyces sp. Q27107. They are closely related in structure to complestatin and possess an oxindolylalanine moiety in place of the tryptophan residue present in complestatin.

Diheteropeptin, a novel substance with TGF-beta-like activity, produced by a fungus, Diheterospora chlamydosporia. II. Physico-chemical properties and structure elucidation.

The structure of diheteropeptin (1), a TGF-beta-like active substance from Diheterospora chlamydosporia Q58044, was determined to be a new cyclotetrapeptide, cyclo[2aminoisobutyryl-(S)-phenylalanyl-(R)-prolyl-(2S,8R,9R)-2-am ino-8,9-dihydroxydecanoyl-] by NMR, mass spectrometric and chemical studies.

Diheteropeptin, a new substance with TGF-beta-like activity, produced by a fungus, Diheterospora chlamydosporia. I. Production, isolation and biological activities.

A new metabolite, diheteropeptin, was found in the culture broth of Diheterospora chlamydosporia Q58044 by screening for TGF-beta-like active substances. Diheteropeptin was extracted from the culture supernatant and purified by a series of chromatographies such as silica gel, gel filtration and HPLC. Diheteropeptin exhibited cytostatic activity in Mv1Lu cells with an IC50 value of 20.3 microM and inhibited histone deacetylase.

Reconstruction of orbicularis oris and levator labii superioris muscles in secondary repair of unilateral cleft lip.

We devised a new method to repair the depression of the nasal floor and inferolateral displacement of the alar base and to reconstruct the philtrum in the secondary repair of unilateral cleft lip. Depression of the nasal floor and inferolateral displacement of the alar base were corrected by advancing a lump of the levator labii superioris, the levator labii superioris alaeque nasi, and the upper part of the superficial orbicularis oris muscles to the anterior nasal spine. When the depression of the nasal floor was too severe to repair using these muscles only, a cranially-based de-epithelialised flap of the scar region on the upper lip was inserted under the nasal floor. The lower, greater part of the superficial orbicularis oris muscle was dissected to the nasolabial fold, brought towards the midline, and laid on the surface of the same muscle on the medial side to be sutured. When the depression of the nasal floor was not severe, the lower, greater part of the superficial orbicularis oris muscle was passed through a tunnel pierced beneath the de-epithelialised scar tissue and sutured to the corresponding components on the medial side to reinforce the philtral ridge. In both cases, if the deep orbicularis oris muscle in the vermilion had been interrupted, it was reconstructed by end-to-end anastomosis. Operative results were evaluated in 76 patients using photographs taken preoperatively and postoperatively. Elevation of the nasal floor and correction of the alar base were achieved in most patients, while reconstruction of the philtrum was achieved in cases in which the skin tension at the suture line was weak.

A novel neuronal cell protecting substance mescengricin produced by Streptomyces griseoflavus.

Neuronal cell death in brain ischemia reperfusion injury such as stroke was induced by L-glutamate toxicity (Choi, D.W. J. Neurosci. 1990. 10, 2493 2501; Coyle, J.T. and Puttfarcken, P. Science 1993, 262, 689 695). In the course of our screening for neuronal cell protecting substances of microbial origin, we isolated a novel compound designated mescengricin from Streptomyces griseoflavus 2853-SVS4(Kim, J.-S., Shin-ya, K., Furihata, K., Hayakawa, Y. and Seto, H. Tetrahedron Lett. 1997. 38, 3431 3434). The structure of mescengricin was determined by a variety of NMR experiments such as HMBC, D-HMBC (Furihata, K., Seto, H. Tetrahedron Lett. 1995. 36, 2817 2820), 1H 15N HMBC (15N-HMBC). It possesses an alpha-carboline structure substituted by a glycerol-ester and a hydroxydihydropyrone. Mescengricin protected chick primary mesencephalic neurons from L-glutamate toxicity with EC50 value 6.0 nM.