TAILIEUCHUNG - Báo cáo khoa học: Onconase cytotoxicity relies on the distribution of its positive charge

Onconase (ONC) is a member of the ribonuclease A superfamily that is toxic to cancer cells in vitro andin vivo. ONC is now in Phase IIIb clinical trials for the treatment of malignant mesothelioma. Internalization of ONC to the cytosol of cancer cells is essential for its cytotoxic activity, despite the apparent absence of a cell-surface receptor protein. | Onconase cytotoxicity relies on the distribution of its positive charge Rebecca F. Turcotte1 Luke D. Lavis2 and Ronald T. Raines2 3 1 MedicalScientist Training Program and Biophysics Graduate Program University of Wisconsin-Madison WI USA 2 Department of Chemistry University of Wisconsin-Madison WI USA 3 Department of Biochemistry University of Wisconsin-Madison WI USA Keywords cancer Coulombic interaction cytotoxin enzyme ribonuclease Correspondence R. T. Raines Department of Biochemistry University of Wisconsin-Madison 433 Babcock Drive Madison WI 53706-1544 USA Fax 1 608 890 2583 Tel 1 608 262 8588 E-mail rtraines@ Website http raines Received 15 December 2008 revised 7 May 2009 accepted 14 May 2009 doi Onconase ONC is a member of the ribonuclease A superfamily that is toxic to cancer cells in vitro and in vivo. ONC is now in Phase Illb clinical trials for the treatment of malignant mesothelioma. Internalization of ONC to the cytosol of cancer cells is essential for its cytotoxic activity despite the apparent absence of a cell-surface receptor protein. Endocytosis and cytotoxicity do however appear to correlate with the net positive charge of ribonucleases. To dissect the contribution made by the endogenous arginine and lysine residues of ONC to its cytotoxicity 22 variants were created in which cationic residues were replaced with alanine. Variants with the same net charge 2 to 5 as well as equivalent catalytic activity and conformational stability were found to exhibit large 10-fold differences in toxicity for the cells of a human leukemia line. In addition a more cationic ONC variant could be either much more or much less cytotoxic than a less cationic variant again depending on the distribution of its cationic residues. The endocytosis of variants with widely divergent cytotoxic activity was quantified by flow cytometry using a small-molecule fluorogen-ic label and was found to vary by twofold or

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