title: Targeting DNA-PKcs and telomerase in brain tumour cells.
abstract: BACKGROUND
  Patients suffering from brain tumours such as glioblastoma and medulloblastoma have poor prognosis with a median survival of less than a year. Identifying alternative molecular targets would enable us to develop different therapeutic strategies for better management of these tumours.
  
  
  METHODS
  Glioblastoma (MO59K and KNS60) and medulloblastoma cells (ONS76) were used in this study. Telomerase inhibitory effects of MST-312, a chemically modified-derivative of epigallocatechin gallate, in the cells were assessed using telomere repeat amplification protocol. Gene expression analysis following MST-312 treatment was done by microarray. Telomere length was measured by telomere restriction fragments analysis. Effects of MST-312 on DNA integrity were evaluated by single cell gel electrophoresis, immunofluorescence assay and cytogenetic analysis. Phosphorylation status of DNA-PKcs was measured with immunoblotting and effects on cell proliferation were monitored with cell titre glow and trypan blue exclusion following dual inhibition.
  
  
  RESULTS
  MST-312 showed strong binding affinity to DNA and displayed reversible telomerase inhibitory effects in brain tumour cells. In addition to the disruption of telomere length maintenance, MST-312 treatment decreased brain tumour cell viability, induced cell cycle arrest and double strand breaks (DSBs). DNA-PKcs activation was observed in telomerase-inhibited cells presumably as a response to DNA damage. Impaired DNA-PKcs in MO59J cells or in MO59K cells treated with DNA-PKcs inhibitor, NU7026, caused a delay in the repair of DSBs. In contrast, MST-312 did not induce DSBs in telomerase negative osteosarcoma cells (U2OS). Combined inhibition of DNA-PKcs and telomerase resulted in an increase in telomere signal-free chromosomal ends in brain tumour cells as well. Interestingly, continual exposure of brain tumour cells to telomerase inhibitor led to population of cells, which displayed resistance to telomerase inhibition-mediated cell arrest. DNA-PKcs ablation in these cells, however, confers higher cell sensitivity to telomerase inhibition, inducing cell death.
  
  
  CONCLUSIONS
  Efficient telomerase inhibition was achieved with acute exposure to MST-312 and this resulted in subtle but significant increase in DSBs. Activation of DNA-PKcs might indicate the requirement of NHEJ pathway in the repair telomerase inhibitor induced DNA damage. Therefore, our results suggest a potential strategy in combating brain tumour cells with dual inhibition of telomerase and NHEJ pathway.
authors.forename: Resham Lal
authors.initials: RL
authors.lastname: Gurung
authors.forename: Hui Kheng
authors.initials: HK
authors.lastname: Lim
authors.forename: Shriram
authors.initials: S
authors.lastname: Venkatesan
authors.forename: Phoebe Su Wen
authors.initials: PS
authors.lastname: Lee
authors.affiliation: Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, 2 Medical Drive, Singapore 117597, Singapore. phsmph@nus.edu.sg.
authors.forename: M Prakash
authors.initials: MP
authors.lastname: Hande
mesh_terms: D001549:Benzamides / Q000494:pharmacology; D001932:Brain Neoplasms / Q000201:enzymology* / Q000235:genetics; D002453:Cell Cycle / Q000187:drug effects; D016923:Cell Death / Q000187:drug effects; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D053903:DNA Breaks, Double-Stranded / Q000187:drug effects; D004273:DNA, Neoplasm / Q000378:metabolism; D051747:DNA-Activated Protein Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D058990:Molecular Targeted Therapy; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D019098:Telomerase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D016615:Telomere / Q000378:metabolism; D059506:Telomere Shortening / Q000187:drug effects
chemical_list: D001549:Benzamides; D004273:DNA, Neoplasm; C470197:MST 312; D012333:RNA, Messenger; D051747:DNA-Activated Protein Kinase; D019098:Telomerase