Plasma membrane nucleolin is a receptor for the anticancer aptamer AS1411 in MV4-11 leukemia cells.
AS1411 is a DNA aptamer that is in phase II clinical trials for relapsed or refractory acute myeloid leukemia and for renal cell carcinoma. AS1411 binds to nucleolin, a protein that is overexpressed in the cytoplasm and on the plasma membrane of some tumor cells compared with normal cells. Studies were performed to determine whether cell surface nucleolin is a receptor for AS1411 in the acute myeloid leukemia cell line MV4-11. Biotinylation of MV4-11 cell surface proteins followed by immunoblotting of the biotinylated proteins showed that full-length (106 kDa) and truncated forms of nucleolin were present on the cell surface. In contrast, K-562 cells, which are 4-fold less sensitive than MV4-11 cells to AS1411, showed no full-length nucleolin and lesser amounts of the truncated forms of nucleolin on the cell surface. Incubation of MV4-11 cells with [(32)P]AS1411 and immunoprecipitation of the plasma membrane fraction with anti-nucleolin antibody demonstrated the presence of [(32)P]AS1411-nucleolin complexes. Anti-nucleolin antibody inhibited binding of fluorescein isothiocyanate (FITC)-AS1411 to plasma membrane nucleolin 56 +/- 10% SE (P < 0.01) compared with cells incubated with FITC-AS1411 only. Cellular uptake of [(32)P]AS1411 into MV4-11 cells was blocked by a 20-fold excess of unlabeled AS1411 but not by a 20-fold excess of the biologically inactive oligonucleotide CRO-26. Uptake was approximately 3-fold faster into MV4-11 cells than into K-562 cells. Partial knockdown of plasma membrane and cytosolic nucleolin in MCF-7 cells resulted in a 3-fold decrease in AS1411 uptake. These results provide evidence that plasma membrane nucleolin is a functional receptor for AS1411 in MV4-11 cells.
Soundararajan
Sridharan
S
Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC 29425, USA.
Wang
Li
L
Sridharan
Vijayalakshmi
V
Chen
Weiwei
W
Courtenay-Luck
Nigel
N
Jones
David
D
Spicer
Eleanor K
EK
Fernandes
Daniel J
DJ
D000818:Animals; D000970:Antineoplastic Agents / Q000378:metabolism* / Q000494:pharmacology / Q000627:therapeutic use; D052157:Aptamers, Nucleotide / Q000378:metabolism* / Q000494:pharmacology / Q000627:therapeutic use; D045744:Cell Line, Tumor; D006801:Humans; D020014:K562 Cells; D015470:Leukemia, Myeloid, Acute / Q000188:drug therapy / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D009838:Oligodeoxyribonucleotides / Q000378:metabolism* / Q000494:pharmacology / Q000627:therapeutic use; D010750:Phosphoproteins / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology; D016601:RNA-Binding Proteins / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000378:metabolism; D000097796:Nucleolin
D000970:Antineoplastic Agents; D052157:Aptamers, Nucleotide; D008565:Membrane Proteins; D009838:Oligodeoxyribonucleotides; D010750:Phosphoproteins; D016601:RNA-Binding Proteins; D011956:Receptors, Cell Surface; D000097796:Nucleolin; C513936:AGRO 100