J Appl Toxicol.2013 Jul;33(7):537-545

Aneugenic potential of the anticancer drugs melphalan and chlorambucil. The involvement of apoptosis and chromosome segregation regulating proteins.

Maria Efthimiou a, Georgia Stephanou a, Nikos A. Demopoulos a and Sotiris S. Nikolaropoulosb

a Division of Genetics, Cell and Developmental Biology, Department of Biology,

and b Laboratory of Medicinal Chemistry, Department of Pharmacy,University of Patras, 26 500, Patras, Greece

 

Abstract

Previous findings showed that the anticancer drugs p-N,N-bis(2-chloroethyl) amino-L-phenylalanine (melphalan, MEL) and p-N,N-bis(2-chloroethyl)aminophenylbutyric acid (chlorambucil, CAB) belonging to the nitrogen mustard group, in addition to their clastogenic activity, also exert aneugenic potential, nondisjunction and chromosome delay. Their aneugenic potential is mainly mediated through centrosome defects. To further investigate their aneugenicity we (a) studied whether apoptosis is a mechanism responsible for the elimination of damaged cells generated by MEL and CAB and (b) investigated if proteins that regulate chromosome segregation are involved in the modulation of their aneugenic potential. Apoptosis was studied by Annexin-V/Propidium Iodide staining and fluorescence microscopy. The involvement of apoptosis on the exclusion of cells with genetic damage and centrosome disturbances was analyzed by DAPI staining and immunofluorescence of b- and g-tubulin in the presence of pan-caspase inhibitor. The expressions of Aurora-A, Aurora-B, survivin and γ-tubulin were studied by western blot. We found that (a) apoptosis is not the mechanism of choice for selectively eliminating cells with supernumerary centrosomes, and (b) the proteins Aurora-A, Aurora-B and survivin are involved in the modulation of MEL and CAB aneugenicity. These findings are important for the understanding of the mechanism responsible for the aneugenic activity of the anticancer drugs melphalan and chlorambucil.

Keywords: anticancer drugs; melphalan and chlorambucil aneugenic potential; apoptosis; supernumerary centrosomes; chromosome segregation regulating proteins

Copyright © 2011 John Wiley & Sons, Ltd.
PMID: 22025197

 

SUPPLEMENT:

We revealed that melphalan and chlorambucil, with known DNA damage activity, also exert aneugenic properties. Their ability to affect chromosome segregation seems to be mediated:  First, by generating cells with abnormal centrosome numbers, which are not eliminated by apoptosis. Abnormalities in centrosome number lead to abnormal mitotic spindles and unequal chromosome distribution to daughter cells. Second, by changing the expression of proteins known to play key role on chromosome segregation during cell division. They reduce the expression of Aurora-B and survivin and enhance the expression of Aurora-A. Aurora kinases play critical roles in genome stability. Aurora-A is required for spindle assembly, whereas Aurora-B for chromosome segregation and cytokinesis.  Survivin inhibits apoptosis and is involved in the regulation of proper chromosome segregation during mitosis. Suppression of survivin, leads to cells with multiple centrosomes. We may say that besides the ability of melphalan and chlorambucil to induce DNA damage, their aneugenic potential is an additional risk that may contribute to the development of secondary tumors in patients under chemotherapy.Figure 1Acknowledgments: This research was partially supported by the Greek State Scholarship’s Foundation to M. E.

Correspondence to: G. Stephanou, Division of Genetics, Cell and Developmental Biology, Department of Biology, University of Patras, 26 500 Patras, Greece. E-mail: geosteph@biology.upatras.gr

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