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

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 β- and γ-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.

Induction of centrosome amplification by formaldehyde, but not hydroquinone, in human lymphoblastoid TK6 cells

“…In our study, melphalan and etoposide induced chromosome gain and breakage in a dose‐dependent manner, at concentrations that produced cytotoxicity, confirming previous findings of aneuploidy and chromosome breakage by melphalan and etoposide in patients with therapy‐related leukemia [Rowley et al, ; Smith et al, ; Pedersen‐Bjergaard et al, ; Pedersen‐Bjergaard et al, ]. Similarly, in our study, centrosome amplification was induced in a dose‐dependent manner by melphalan and etoposide, confirming it as a potential mechanism underlying leukemogenesis associated with these leukemogens [Shimada et al, ; Efthimiou et al, ].…”

“…Here, we sought to determine whether FA and HQ, which have been shown to induce aneuploidy in human blood cells [Zhang et al, 2005, 2010b], could induce centrosome amplification in TK6 cells. We included melphalan and etoposide as positive controls for centrosome amplification [Efthimiou et al, 2013]. We examined centrosome amplification, chromosomal gain, loss, and breakage in TK6 cells that were treated with several concentrations doses of each chemical for 24 h, washed and allowed to recover for 24 h.…”

“…Kumari et al previously reported that high levels of FA (300 μM) induced aneuploidy, polyploidy and abnormal centrosome defects in Chinese hamster ovary (CHO) cells [Kumari et al, 2012]. We included melphalan and etoposide as positive controls in the study as they were previously reported to induce centrosome amplification in human lymphocytes [Efthimiou et al, 2013] and human U2OS and mouse NIH3T3 cells [Shimada et al, 2011] in vitro, respectively. We investigated centrosome amplification, chromosome aberrations (aneuploidy and breakage) and cytotoxicity induced by treatment with several concentrations of each chemical for 24 h, followed by a 24-h recovery period.…”

DNA Damage and Repair in Human Cancer: Molecular Mechanisms and Contribution to Therapy-Related Leukemias

“…Thus an imbalance in some of the components of the repair pathways might be responsible for inefficient DNA repair and accumulation of mutations contributing to the development of t-MN. In addition to the acknowledged clastogenicity of these drugs, attention on their aneugenic potential possibly because of the ability to induce centrosome defects, has also been raised [ 47 ].…”

Combined study on clastogenic, aneugenic and apoptotic properties of doxorubicin in human cells in vitro

“…The protein amount in the bands of each lane was quantified by densitometric analysis relative to the actin numerical quantities. In each independent experiment, protein amount variation fold was calculated considering equal to 1 the protein/β-actin ratio observed in untreated cells [ 25 , 26 ].…”