title: 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.
abstract: BACKGROUND
  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. 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.
  
  
  METHODS
  Normal 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.
  
  
  RESULTS
  Here 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.
  
  
  CONCLUSIONS
  Our 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.
authors.affiliation: Department of Clinical and Biological Sciences, University of Turin, 10043 Orbassano, Italy. valentina.turinetto@unito.it
authors.forename: Valentina
authors.initials: V
authors.lastname: Turinetto
authors.forename: Paola
authors.initials: P
authors.lastname: Porcedda
authors.forename: Luca
authors.initials: L
authors.lastname: Orlando
authors.forename: Mario
authors.initials: M
authors.lastname: De Marchi
authors.forename: Antonio
authors.initials: A
authors.lastname: Amoroso
authors.forename: Claudia
authors.initials: C
authors.lastname: Giachino
mesh_terms: D017209:Apoptosis / Q000187:drug effects*; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors*; D004261:DNA Replication / Q000187:drug effects*; D004004:Dichlororibofuranosylbenzimidazole / Q000494:pharmacology*; D006801:Humans; D007938:Leukemia / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism / Q000503:physiopathology; D008214:Lymphocytes / Q000166:cytology / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000493:pharmacokinetics*; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*
chemical_list: D047428:Protein Kinase Inhibitors; D016159:Tumor Suppressor Protein p53; D004004:Dichlororibofuranosylbenzimidazole; D018844:Cyclin-Dependent Kinases
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