The cyclin-dependent kinase inhibitor 5, 6-dichloro-1-beta-D-ribofuranosylbenzimidazole induces nongenotoxic, DNA replication-independent apoptosis of normal and leukemic cells, regardless of their p53 status

Valentina Turinetto,Luca Orlando,Antonio Amoroso,Mario De Marchi,Claudia Giachino,Paola Porcedda

doi:10.1186/1471-2407-9-281

Abstract

BackgroundCurrent chemotherapy of human cancers focuses on the DNA damage pathway to induce a p53-mediated cellular response leading to either G1 arrest or apoptosis. However, genotoxic treatments may induce mutations and translocations that result in secondary malignancies or recurrent disease. In addition, about 50% of human cancers are associated with mutations in the p53 gene. Nongenotoxic activation of apoptosis by targeting specific molecular pathways thus provides an attractive therapeutic approach.MethodsNormal and leukemic cells were evaluated for their sensitivity to 5, 6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) through cell viability and caspase activation tests. The apoptotic pathway induced by DRB was analysed by immunfluorescence and immunoblot analysis. H2AX phosphorylation and cell cycle analysis were performed to study the dependance of apoptosis on DNA damage and DNA replication, respectively. To investigate the role of p53 in DRB-induced apoptosis, specific p53 inhibitors were used. Statistical analysis on cell survival was performed with the test of independence.ResultsHere we report that DRB, an inhibitor of the transcriptional cyclin-dependent kinases (CDKs) 7 and 9, triggers DNA replication-independent apoptosis in normal and leukemic human cells regardless of their p53 status and without inducing DNA damage. Our data indicate that (i) in p53-competent cells, apoptosis induced by DRB relies on a cytosolic accumulation of p53 and subsequent Bax activation, (ii) in the absence of p53, it may rely on p73, and (iii) it is independent of ATM and NBS1 proteins. Notably, even apoptosis-resistant leukemic cells such as Raji were sensitive to DRB.ConclusionOur results indicate that DRB represents a potentially useful cancer chemotherapeutic strategy that employs both the p53-dependent and -independent apoptotic pathways without inducing genotoxic stress, thereby decreasing the risk of secondary malignancies.

Highlights

Current chemotherapy of human cancers focuses on the DNA damage pathway to induce a p53-mediated cellular response leading to either G1 arrest or apoptosis
The stress response elicited by DRB in human lymphocytes results in DNA replication-independent, DNA damageindependent and p53-mediated apoptosis We investigated the effects induced by DRB in human cells
To define the sensitivity of cultured T lymphocytes to this drug, we assessed the viability of treated cells in a dose-response experiment using propidium iodide (PI) staining and flow cytometry

Summary

Introduction

Current chemotherapy of human cancers focuses on the DNA damage pathway to induce a p53-mediated cellular response leading to either G1 arrest or apoptosis. Genotoxic treatments may induce mutations and translocations that result in secondary malignancies or recurrent disease. Current chemotherapy focuses on the use of genotoxic drugs This may induce general DNA damage in cancer cells and high levels of toxicity in normal tissues. Radiotherapy, damage tumour cell DNA to induce arrest in G1 or apoptosis [5,6]. Most chemotherapy treatments rely on induction of p53-dependent apoptosis The efficiency of this approach, is diminished by the fact that the p53 gene is mutated in about 50% of human cancers. It is becoming clear that a high percentage of resistant and recurrent tumours carry de novo p53 mutations [2,4,6,10]

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