Takanori Kubo

Antisense inhibition of Bcr-Abl/c-Abl synthesis promotes telomerase activity and upregulates tankyrase in human leukemia cells1

Clinical studies in chronic myelogenous leukemia demonstrate that the overexpression of Bcr‐Abl tyrosine kinase is usually accompanied by relatively low telomerase activity in the chronic phase, which reverts to a high activity in blast crisis. The present study was designed to investigate the cross‐talk between both enzymes, using Bcr‐Abl‐positive K‐562 and Bcr‐Abl‐negative Jurkat cell lines, treated with antisense oligodeoxyribonucleotides (ODNs) against Bcr‐Abl/c‐Abl mRNA. The decreased amount and enzyme activity of Bcr‐Abl/c‐Abl provoked telomerase activation in both cell lines. After short‐term treatment with anti‐Bcr‐Abl/c‐Abl ODNs (6 days), no variations in hTERT and phospho‐hTERT were detected. The decreased amount of Bcr‐Abl/c‐Abl was accompanied by: alterations in telomeric associated proteins–overexpression of tankyrase and decreased amount of TRF1/Tin2, cell growth arrest of K‐562 cells, reaching a plateau after 6 days treatment, and increased proliferating activity of Jurkat cells. No changes in telomere length were detected after short‐term treatment. In contrast, after long‐term treatment with anti‐Bcr‐Abl/c‐Abl ODNs (36 days), a significant elongation of telomeres and enhancement of hTERT were established, accompanied by an increased proliferating activity of both cell lines. These data provide evidence that the inhibition of Bcr‐Abl or c‐Abl synthesis keeps a potential to restore or induce cell proliferation through telomere lengthening control and telomerase activation.

SPECIFIC BINDING AND STABILIZATION OF DNA AND PHOSPHOROTHIOATE DNA BY AMPHIPHILIC α-HELICAL PEPTIDES

Synthetic cationic peptides with amphiphilic α-helical structure were found to have DNA binding ability to stabilize double and triple stranded DNA. The stabilization effect was significant for cationic α-helical peptides indicating the importance of an electrostatic interaction of a positive charge of peptides and a negative charge of DNAs.

A NOVEL APPROACH FOR THE SOLID PHASE SYNTHESIS OF DNA-PEPTIDE CONJUGATES

The strategy of this study involves automated synthesis of oligonucleotides on a CPG support using standard cyanoethyl phosphoramidite chemistry followed by covalent linkage to peptide fragments bearing a free terminal α-amino group and residues with protected side chains. Conjugation was formed through an alkyldiisocyanate linker. Conjugates wereisolated by cleavage from the solid support and deprotection in one step.

AMPHIPHILIC β-SHEET PEPTIDES CAN BIND TO DOUBLE AND TRIPLE STRANDED DNA

It was shown that synthetic peptides with amphiphilic β-sheet structure can bind to and stabilize double and triple stranded DNA. CD spectra indicated that β-sheet conformation of peptides were emphasized in the presence or absence of DNA and that no significant change of DNA conformation occurred. UV melting study at pH 7.0 revealed that interaction of peptides with DNA and its hybrids are sensitive and specific depending the host structure.

Control of intracellular delivery of oligonucleotides by conjugation with signal peptides.

It has been shown that intracellular localization of proteins are controlled by the interaction between signal domains in proteins to transport and proteins to be transported. Taira and his colleagues have reported that efficiency of ribozymes largely depended on the intracellular localization of it. In the present paper, rational control of intracellular delivery and localization of oligonucleotides by oligonucleotide-signal peptide conjugates was studied.

DNA Conjugates as Novel Functional Oligonucleotides

Abstract Oligodeoxynucleotides with RNA cleavage activity 1) were conjugated with amines and peptides by solid phase fragment condensation (SPFC). It was found that 29 mer DNA enzyme conjugated with spermine at its 5′-end showed higher affinity to the target RNA sequence and 40 times higher activity of cleavage than native DNA enzyme. It is also to be noted that conjugate DNA enzymes showed increased resistance against nuclease digestion

Synthesis of DNA Conjugates by Solid Phase Fragment Condensation

Abstract Development of a novel method for the synthesis of DNA conjugates is described. Oligonucleotides were successfully conjugated with a variety of functional molecules on a solid phase (Solid Phase Fragment Condensation) using an amino, a hydroxyl, a thiol, and a carboxyl group. DNA-peptide conjugate was obtained as a pure from by a single RPHPLC purification approximately in 20% yield. Moreover, it was demonstrated that the present method was effective for the preparation of conjugate molecules, DNA-sugar, DNA-polyamine, DNA-lipid and so on. The study to create new intelligent DNAs by accumulation various biofunctions on the molecule by SPFC is now in progress in our laboratory.

Novel synthesis of 2'-O modified oligonucleotides by solid phase fragment condensation.

Oligonucleotides modified at 20-OH position have attracted much attention from a biological and medicinal point of view because they have a number of advantageous properties for their application to antisense and antigene medicines. For example, they can bind to complementary RNA with higher affinity than native oligoDNA, and they are more resistant to nuclease degradation. It has been shown by the researchers of ISIS Pharmaceuticals Ltd. that ‘‘gapmer technology’’ combining this type of modification and sulfurization of phosphate backbone of oligonucleotides makes it possible to apply chemically modified oligonucleotides to antisense therapy.