Oligodeoxynucleotide 5mers containing a 5'-CpG induce apoptosis through a mitochondrial mechanism in T lymphocytic leukaemia cells.
A chimeric methylphosphonodiester/phosphodiester 15mer oligodeoxynucleotide of randomly selected sequence was observed to rapidly induce apoptosis in MOLT-4 and Jurkat E6 T lymphocytic leukaemia cells following intracytoplasmic delivery. A series of further methylphosphonate substitutions and mutations and truncations of the oligodeoxynucleotide served to establish that the phosphodiester-linked sequence CGGTA present in the 15mer was responsible for this biological activity. End-protected CpG oligodeoxy-nucleotide 5mers of sequence type CGNNN exhibited a range of apoptosis-inducing potencies, with CGTTA being the most active. The latter was shown to significantly reduce the rate of RNA synthesis in MOLT-4 cells within 1 h; DNA laddering and redistribution of phosphatidylserine to the outer surface of the plasma membrane were marked by 160 min and mitochondrial transmembrane potential collapsed over roughly the same time scale. Pro-caspase 8 was reduced within 130 min and the proteolytically activated caspase 8 substrate Bid was also down by this time, implicating release of cytochrome c from mitochondria by the active 15 kDa fragment of Bid. Substantial proteolytic activation of pro-caspase 3 was relatively delayed. These findings support a mitochondrial amplification mechanism for apoptosis triggered by CpG 5mers.
Tidd
D M
DM
School of Biological Sciences and Department of Haematology, The University of Liverpool, UK. dmtidd@liv.ac.uk
Spiller
D G
DG
Broughton
C M
CM
Norbury
L C
LC
Clark
R E
RE
Giles
R V
RV
D017209:Apoptosis / Q000235:genetics*; D051023:BH3 Interacting Domain Death Agonist Protein; D002352:Carrier Proteins / Q000378:metabolism; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D018899:CpG Islands; D004792:Enzyme Precursors / Q000378:metabolism; D006801:Humans; D019169:Jurkat Cells; D007945:Leukemia, Lymphoid; D008564:Membrane Potentials; D008928:Mitochondria / Q000378:metabolism; D009838:Oligodeoxyribonucleotides / Q000494:pharmacology*; D010718:Phosphatidylserines; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism; D014407:Tumor Cells, Cultured; D014529:Uridine / Q000378:metabolism
D051023:BH3 Interacting Domain Death Agonist Protein; C494814:BID protein, human; D002352:Carrier Proteins; D004792:Enzyme Precursors; D009838:Oligodeoxyribonucleotides; D010718:Phosphatidylserines; D016271:Proto-Oncogene Proteins c-myc; D012333:RNA, Messenger; C505530:CASP8 protein, human; C506433:CASP9 protein, human; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases; D014529:Uridine