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Cytotoxic G-rich oligodeoxynucleotides: putative protein targets and required sequence motif.

Goodchild A, King A, Gozar MM, Passioura T, Tucker C, Rivory L - Nucleic Acids Res. (2007)

Bottom Line: Nucleolin, elongation factor 1-alpha (eEF1A) and vimentin were identified as binding to a cytotoxic ODN (Dz13) using protein pull-down assays and LC-MS/MS.Although these proteins have previously been described to bind G-rich ODNs, the binding of eEF1A correlated with cytotoxicity, whereas binding of nucleolin and vimentin did not.Although the exact mechanism of cytotoxicity remains unknown, marked potency of the longer (> or =25 nt) ODNs in particular, indicates the potential of these molecules for treatment of diseases associated with abnormal cell proliferation.

View Article: PubMed Central - PubMed

Affiliation: Johnson & Johnson Research Pty Ltd, Eveleigh, NSW, 1430, Australia. agoodchi@medau.jnj.com

ABSTRACT
It has recently been shown that certain oligodeoxynucleotides (ODNs) designed as catalytic DNA molecules (DNAzymes) exhibit potent cytotoxicity independent of RNA-cleavage activity in a number of cell lines. These cytotoxic ODNs all featured a 5' G-rich sequence and induced cell death by a TLR9-independent mechanism. In this study, we examined the sequence and length dependence of ODNs for cytotoxicity. A G-rich sequence at the 5' terminus of the molecule was necessary for cytotoxicity and the potency of ODNs with active 5' sequences was length dependent. Cytotoxicity appeared to be generally independent of 3' sequence composition, although 3' sequences totally lacking G-nucleotides were mostly inactive. Nucleolin, elongation factor 1-alpha (eEF1A) and vimentin were identified as binding to a cytotoxic ODN (Dz13) using protein pull-down assays and LC-MS/MS. Although these proteins have previously been described to bind G-rich ODNs, the binding of eEF1A correlated with cytotoxicity, whereas binding of nucleolin and vimentin did not. Quiescent non-proliferating cells were resistant to cytotoxicity, indicating cytotoxicity may be cell cycle dependent. Although the exact mechanism of cytotoxicity remains unknown, marked potency of the longer (> or =25 nt) ODNs in particular, indicates the potential of these molecules for treatment of diseases associated with abnormal cell proliferation.

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Binding of eEF1A from different cellular fractions of HMEC-1 cells. Protein pull-downs using Dz13-beads were performed on cytoplasmic, nuclear and membrane/organelle fractions. 1, eEF1A bound to Dz13-beads; 2, eEF1A eluting from Dz13 beads with 1 μM Dz13; 3, eEF1A eluting from Dz13-beads protein with 1 μM Dz13scr.
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Figure 5: Binding of eEF1A from different cellular fractions of HMEC-1 cells. Protein pull-downs using Dz13-beads were performed on cytoplasmic, nuclear and membrane/organelle fractions. 1, eEF1A bound to Dz13-beads; 2, eEF1A eluting from Dz13 beads with 1 μM Dz13; 3, eEF1A eluting from Dz13-beads protein with 1 μM Dz13scr.

Mentions: To determine the cellular localization of the eluting eEF1A, the pull-down procedure was performed on the cytoplasmic, nuclear, membrane and cytoskeletal fractions of HMEC-1 cells. Whereas eEF1A from the cytoplasmic, nuclear and membrane/organelle fractions bound to Dz13 beads, the major source of eEF1A eluted by Dz13 was from the cytoplasm (Figure 5). Pull-downs were also performed with total protein lysates from a number of other cell lines including RSMC, HEK-293 and HCT-116, to which Dz13 is cytotoxic (15). Predominant elution by Dz13 of the 51 kDa band was observed in all cases and this band was confirmed as being eEF1A by western blotting (data not shown). The results indicate that eEF1A binding also occurs in other cell lines in which Dz13 is cytotoxic. When examined directly by western blotting, the abundance of eEF1A was not dependent on the cell type and was not affected by treatment with Dz13 for 24 h (data not shown).Figure 5.


Cytotoxic G-rich oligodeoxynucleotides: putative protein targets and required sequence motif.

Goodchild A, King A, Gozar MM, Passioura T, Tucker C, Rivory L - Nucleic Acids Res. (2007)

Binding of eEF1A from different cellular fractions of HMEC-1 cells. Protein pull-downs using Dz13-beads were performed on cytoplasmic, nuclear and membrane/organelle fractions. 1, eEF1A bound to Dz13-beads; 2, eEF1A eluting from Dz13 beads with 1 μM Dz13; 3, eEF1A eluting from Dz13-beads protein with 1 μM Dz13scr.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1935016&req=5

Figure 5: Binding of eEF1A from different cellular fractions of HMEC-1 cells. Protein pull-downs using Dz13-beads were performed on cytoplasmic, nuclear and membrane/organelle fractions. 1, eEF1A bound to Dz13-beads; 2, eEF1A eluting from Dz13 beads with 1 μM Dz13; 3, eEF1A eluting from Dz13-beads protein with 1 μM Dz13scr.
Mentions: To determine the cellular localization of the eluting eEF1A, the pull-down procedure was performed on the cytoplasmic, nuclear, membrane and cytoskeletal fractions of HMEC-1 cells. Whereas eEF1A from the cytoplasmic, nuclear and membrane/organelle fractions bound to Dz13 beads, the major source of eEF1A eluted by Dz13 was from the cytoplasm (Figure 5). Pull-downs were also performed with total protein lysates from a number of other cell lines including RSMC, HEK-293 and HCT-116, to which Dz13 is cytotoxic (15). Predominant elution by Dz13 of the 51 kDa band was observed in all cases and this band was confirmed as being eEF1A by western blotting (data not shown). The results indicate that eEF1A binding also occurs in other cell lines in which Dz13 is cytotoxic. When examined directly by western blotting, the abundance of eEF1A was not dependent on the cell type and was not affected by treatment with Dz13 for 24 h (data not shown).Figure 5.

Bottom Line: Nucleolin, elongation factor 1-alpha (eEF1A) and vimentin were identified as binding to a cytotoxic ODN (Dz13) using protein pull-down assays and LC-MS/MS.Although these proteins have previously been described to bind G-rich ODNs, the binding of eEF1A correlated with cytotoxicity, whereas binding of nucleolin and vimentin did not.Although the exact mechanism of cytotoxicity remains unknown, marked potency of the longer (> or =25 nt) ODNs in particular, indicates the potential of these molecules for treatment of diseases associated with abnormal cell proliferation.

View Article: PubMed Central - PubMed

Affiliation: Johnson & Johnson Research Pty Ltd, Eveleigh, NSW, 1430, Australia. agoodchi@medau.jnj.com

ABSTRACT
It has recently been shown that certain oligodeoxynucleotides (ODNs) designed as catalytic DNA molecules (DNAzymes) exhibit potent cytotoxicity independent of RNA-cleavage activity in a number of cell lines. These cytotoxic ODNs all featured a 5' G-rich sequence and induced cell death by a TLR9-independent mechanism. In this study, we examined the sequence and length dependence of ODNs for cytotoxicity. A G-rich sequence at the 5' terminus of the molecule was necessary for cytotoxicity and the potency of ODNs with active 5' sequences was length dependent. Cytotoxicity appeared to be generally independent of 3' sequence composition, although 3' sequences totally lacking G-nucleotides were mostly inactive. Nucleolin, elongation factor 1-alpha (eEF1A) and vimentin were identified as binding to a cytotoxic ODN (Dz13) using protein pull-down assays and LC-MS/MS. Although these proteins have previously been described to bind G-rich ODNs, the binding of eEF1A correlated with cytotoxicity, whereas binding of nucleolin and vimentin did not. Quiescent non-proliferating cells were resistant to cytotoxicity, indicating cytotoxicity may be cell cycle dependent. Although the exact mechanism of cytotoxicity remains unknown, marked potency of the longer (> or =25 nt) ODNs in particular, indicates the potential of these molecules for treatment of diseases associated with abnormal cell proliferation.

Show MeSH
Related in: MedlinePlus