Inhibition of DNA Replication and Induction of S Phase Cell Cycle Arrest by G-rich Oligonucleotides

Abstract

The discovery of G-rich oligonucleotides (GROs) that have non-antisense antiproliferative activity against a number of cancer cell lines has been recently described. This biological activity of GROs was found to be associated with their ability to form stable G-quartet-containing structures and their binding to a specific cellular protein, most likely nucleolin (Bates, P. J., Kahlon, J. B., Thomas, S. D., Trent, J. O., and Miller, D. M. (1999) J. Biol. Chem. 274, 26369 -26377). In this report, we further investigate the novel mechanism of GRO activity by examining their effects on cell cycle progression and on nucleic acid and protein biosynthesis. Cell cycle analysis of several tumor cell lines showed that cells accumulate in S phase in response to treatment with an active GRO. Analysis of 5-bromodeoxyuridine incorporation by these cells indicated the absence of de novo DNA synthesis, suggesting an arrest of the cell cycle predominantly in S phase. At the same time point, RNA and protein synthesis were found to be ongoing, indicating that arrest of DNA replication is a primary event in GRO-mediated inhibition of proliferation. This specific blockade of DNA replication eventually resulted in altered cell morphology and induction of apoptosis. To characterize further GRO-mediated inhibition of DNA replication, we used an in vitro assay based on replication of SV40 DNA. GROs were found to be capable of inhibiting DNA replication in the in vitro assay, and this activity was correlated to their antiproliferative effects. Furthermore, the effect of GROs on DNA replication in this assay was related to their inhibition of SV40 large T antigen helicase activity. The data presented suggest that the antiproliferative activity of GROs is a direct result of their inhibition of DNA replication, which may result from modulation of a replicative helicase activity.Oligonucleotides can recognize both nucleic acids and proteins with a high degree of specificity. This is a major reason why they have been widely investigated as potential therapeutic agents for cancer, viral infections, and inflammatory diseases. Oligonucleotides can achieve target recognition by sequence-specific interactions with nucleic acids or proteins such as in the antisense, antigene, or decoy approaches (1-4). Alternatively, target recognition can be due to the specific threedimensional structure of an oligonucleotide, as in the aptamer approach (5, 6). These aptameric oligonucleotides often contain secondary structure elements such as hairpins or G-quartets. The formation of G-quartet structures is also thought to contribute to non-antisense growth inhibitory effects of G-rich phosphodiester and phosphorothioate oligonucleotides (7-9).Recently, we reported (9) on a novel class of phosphodiester G-rich oligonucleotides (GROs) 1 that could strongly inhibit the in vitro proliferation of tumor cells derived from prostate, breast, and cervical carcinomas. The antiproliferative GROs were able to form stable secondary structures consistent with G-quartet format...

PNAS-4, an Early DNA Damage Response Gene, Induces S Phase Arrest and Apoptosis by Activating Checkpoint Kinases in Lung Cancer Cells

“…Synchronization of Cells-Cells were synchronized using the serum starvation method (16). Briefly, A549 and Calu-1 cells were plated in complete medium (DMEM or RPMI 1640) containing 10% FBS, at a density of 5 ϫ 10 4 cells/well in 6-well plates and incubated overnight.…”

Apoptosis in Leukemia Cells Is Accompanied by Alterations in the Levels and Localization of Nucleolin

“…Slides were washed with PBS and incubated in permeabilization solution (0.1% Triton X-100, 0.1% sodium citrate) for 2 min on ice, washed twice with PBS, and dried. Samples were stained as previously described (29). Briefly, 50 l of TUNEL reaction mixture (Roche) was added to each sample and slides were incubated in the dark in a humidified chamber for 60 min at 37°C, then washed three times with PBS and observed as above.…”

Effects of Pro-Arg, a Novel Dipeptide Derived from Protamine Hydrolysate on H2O2-Induced Oxidative Stress in Human Diploid Fibroblasts

“…GRO showed an arrest effect of the cell cycle predominantly in S phase. 38) Joe et al found resveratrol caused an increase of cells in the S phase and a corresponding decrease of cells in the G 1 and G 2 -M phases in HCE7, MCF7, SW480, and HL60 cells. But in Bic-1 cell, resveratrol treatment led to G 1 -phase arrest, and resveratrol did not appear to alter the cell cycle distribution in Seg-1 cell.…”