Antiproliferative effect in chronic myeloid leukaemia cells by antisense peptide nucleic acids.
Peptide nucleic acid (PNA) is a synthetic DNA analogue that is resistant to nucleases and proteases and binds with exceptional affinity to RNA. Because of these properties PNA has the potential to become a powerful therapeutic agent to be used in vivo. Until now, however, the use of PNA in vivo has not been much investigated. Here, we have attempted to reduce the expression of the bcr/abl oncogene in chronic myeloid leukaemia KYO-1 cells using a 13mer PNA sequence (asPNA) designed to hybridise to the b2a2 junction of bcr/abl mRNA. To enhance cellular uptake asPNA was covalently linked to the basic peptide VKRKKKP (NLS-asPNA). Moreover, to investigate the cellular uptake by confocal microscopy, both PNAs were linked by their N-terminus to fluorescein (FL). Studies of uptake, carried out at 4 and 37 degrees C on living KYO-1 cells stained with hexidium iodide, showed that both NLS-asPNA-FL and asPNA-FL were taken up by the cells, through a receptor-independent mechanism. The intracellular amount of NLS-asPNA-FL was about two to three times higher than that of asPNA-FL. Using a semi-quantitative RT- PCR technique we found that 10 micro M asPNA and NLS-asPNA reduced the level of b2a2 mRNA in KYO-1 cells to 20 +/- 5% and 60 +/- 10% of the control, respectively. Western blot analysis showed that asPNA promoted a significant inhibition of p210(BCR/ABL) protein: residual protein measured in cells exposed for 48 h to asPNA was approximately 35% of the control. Additionally, asPNA impaired cell growth to 50 +/- 5% of the control and inhibited completion of the cell cycle. In summary, these results demonstrate that a PNA 13mer is taken up by KYO-1 cells and is capable of producing a significant and specific down-regulation of the bcr/abl oncogene involved in leukaemogenesis.
Department of Biomedical Sciences and Technologies, School of Medicine, University of Udine, Piazzale Kolbe 4, 33100 Udine, Italy.
Valentina
V
Rapozzi
Brigitte E A
BE
Burm
Susanna
S
Cogoi
Gijs A
GA
van der Marel
Jacques H
JH
van Boom
Franco
F
Quadrifoglio
Luigi E
LE
Xodo
D000970:Antineoplastic Agents / Q000378:metabolism / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D001483:Base Sequence; D002453:Cell Cycle / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D016373:DNA, Antisense / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D015536:Down-Regulation; D005434:Flow Cytometry; D019793:Fluorescein / Q000737:chemistry; D016044:Fusion Proteins, bcr-abl / Q000235:genetics; D006801:Humans; D020014:K562 Cells; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000188:drug therapy* / Q000235:genetics / Q000473:pathology; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D019913:Nuclear Localization Signals / Q000235:genetics; D020135:Peptide Nucleic Acids / Q000235:genetics*; D012333:RNA, Messenger / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000378:metabolism
D000970:Antineoplastic Agents; D016373:DNA, Antisense; D019913:Nuclear Localization Signals; D020135:Peptide Nucleic Acids; D012333:RNA, Messenger; D016044:Fusion Proteins, bcr-abl; D019793:Fluorescein