Deoxyribonucleic acid triplex formation inhibits granulocyte macrophage colony-stimulating factor gene expression and suppresses growth in juvenile myelomonocytic leukemic cells.
Juvenile myelomonocytic leukemia (JMML) is a severe childhood malignancy. The autocrine production of GMCSF is believed to be responsible for the spontaneous proliferation of JMML cells. A nuclear factor-kappaB (NF-kappaB)/Rel binding site within the GM-CSF gene promoter, termed the kappaB element, plays an important role in controlling transcription from the GM-CSF gene. We investigated the effect of an oligonucleotide GM3, directed to form a DNA triple helix across this kappaB element, on growth and GM-CSF production by JMML cells. Treatment of these cells, either unstimulated or induced by TNFalpha, with GM3 led to a significant and specific inhibition of both GM-CSF production and spontaneous colony formation. This constitutes the first report linking specific triplex-mediated inhibition of gene transcription with a functional outcome; i.e., cell growth. We observed the constitutive presence of NF-kappaB/Rel proteins in the nucleus of JMML cells. The constitutive and TNFalpha-induced NF-kappaB/Rel complexes were identical and were composed mainly of p50 and c-Rel proteins. Treatment of the cells with a neutralizing anti-TNFalpha monoclonal antibody completely abrogated constitutive nuclear expression of NF-kappaB/Rel proteins. These results indicate that the aberrant, constitutive GM-CSF gene activation in JMML is maintained by TNFalpha-mediated activation of NF-kappaB/Rel proteins. Our findings identify the molecular basis for the autocrine TNFalpha activation of the GM-CSF gene in JMML and suggest potential novel and specific approaches for the treatment of this aggressive childhood leukemia.
Kochetkova
M
M
Division of Human Immunology, Hanson Centre for Cancer Research, Institute of Medical and Veterinary Science, Adelaide, 5000 South Australia, Australia.
Iversen
P O
PO
Lopez
A F
AF
Shannon
M F
MF
D001665:Binding Sites; D002455:Cell Division; D002467:Cell Nucleus / Q000737:chemistry; D002675:Child, Preschool; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D015870:Gene Expression; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics*; D006801:Humans; D007223:Infant; D015477:Leukemia, Myelomonocytic, Chronic / Q000235:genetics / Q000473:pathology*; D016328:NF-kappa B / Q000032:analysis / Q000378:metabolism; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D016329:Sp1 Transcription Factor / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D016328:NF-kappa B; D009838:Oligodeoxyribonucleotides; D016329:Sp1 Transcription Factor; D014409:Tumor Necrosis Factor-alpha; C051802:triplex DNA; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D004247:DNA