Multigene targeting with antisense oligodeoxynucleotides: an exploratory study using primary human leukemia cells.
OBJECTIVE
We previously reported that the c-myb and Vav proto-oncogenes are amenable to silencing with antisense oligodeoxynucleotides and that inhibition of either impairs leukemic cell growth. Because the expression of these genes is not known to be linked, we sought to determine the therapeutic value of silencing both genes simultaneously in K562 and primary patient (n = 9) chronic myelogenous leukemia cells.


METHODS
K562 and primary chronic myelogenous leukemia cells were exposed to antisense oligodeoxynucleotides (alone or in combination) for 24 or 72 hours and then cloned in methylcellulose cultures. Effects on K562 cluster, and blast-forming unit-erythroid colonies and granulocyte-macrophage colony-forming units were determined and correlated with the ability to down-regulate the targeted mRNA.


RESULTS
After 24-hour exposure, K562 cell growth was inhibited in a sequence specific, dose-responsive manner with either c-myb or Vav antisense oligodeoxynucleotides. Exposure to both oligodeoxynucleotides simultaneously considerably enhanced growth inhibition and accelerated apoptosis. Primary cell results were more complex. After 24- and 72-hour exposures to either anti-vav or anti-myb antisense oligodeoxynucleotides, equivalent colony-forming unit inhibition was observed. Exposing cells to both antisense oligodeoxynucleotides simultaneously for 24 hours did not result in additional inhibition of colony formation. However, after 72-hour incubation with both oligodeoxynucleotides, colony formation was diminished significantly when compared with either oligodeoxynucleotides alone (from approximately 30% to approximately 78% for granulocyte-macrophage colony-forming unit; approximately 50% to approximately 80% for blast-forming unit-erythroid).


CONCLUSIONS
We hypothesize that exposing primary leukemic cells to antisense oligodeoxynucleotides targeted to two, or possibly more, genes might significantly augment the therapeutic utility of these molecules.
Opalinska
Joanna B
JB
Authors' Affiliations: Hematology and Pathology, Pommeranian Medical University, Szczecin, Poland.
Machalinski
Boguslaw
B
Ratajczak
Janina
J
Ratajczak
Mariusz Z
MZ
Gewirtz
Alan M
AM
D017209:Apoptosis / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects*; D004305:Dose-Response Relationship, Drug; D004357:Drug Synergism; D006801:Humans; D020014:K562 Cells; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000235:genetics / Q000473:pathology; D014411:Neoplastic Stem Cells / Q000187:drug effects; D020319:Oligodeoxyribonucleotides, Antisense / Q000235:genetics / Q000494:pharmacology*; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D014407:Tumor Cells, Cultured; D014410:Tumor Stem Cell Assay
C489427:MYC protein, human; D020319:Oligodeoxyribonucleotides, Antisense; D016271:Proto-Oncogene Proteins c-myc; D012333:RNA, Messenger