Comparative study of genetic activity of chlorambucil's active metabolite steroidal esters: The role of steroidal skeleton on aneugenic potential

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

“…To study the involvement of apoptosis in the exclusion of cells with mitotic spindle disturbances, C 2 C 12 cells were grown at a density of 1.8 × 10 5 cells ml −1 on 22 × 22 mm glass coveslips in 35 mm Petri dishes and treated with MEL, CAB and PHE at 10 µ m in the absence or presence of 50 µ m Z‐VAD‐FMK for 48 h. According to our previous findings the mitotic index was reduced at about 50% when C 2 C 12 cells were treated with the 10 µ m of the studied compounds. At this concentration MEL and CAB, besides clastogenic activity, also exhibited aneugenic potential owing to centrosome defects (Efthimiou et al ., ; Efthimiou, ). The study of mitotic spindle morphology was performed by fluorescence double staining for β ‐ and γ ‐tubulin as previously described (Efthimiou et al ., ).…”

“…The C 2 C 12 cell line (DSMZ, Germany) was used in this study (Yaffe and Saxel, ; Blau et al ., ). C 2 C 12 mouse cells have the ability to form an extensive microtubule network and comprise a good biological system to study alterations on the organization of mitotic apparatus by treating the cells with the studied compounds (Efthimiou et al ., ). Untreated cells have a near tetraploid karyotype.…”

“…At this concentration MEL and CAB, besides clastogenic activity, also exhibited aneugenic potential owing to centrosome defects (Efthimiou et al ., ; Efthimiou, ). The study of mitotic spindle morphology was performed by fluorescence double staining for β ‐ and γ ‐tubulin as previously described (Efthimiou et al ., ). Fluorescein Isothiocyanate (FITC) and Tetramethylrhodamine (TRITC) fluorophores were used to label centrosomes and microtubules, respectively.…”

“…() showed that two steroidal analogs of PHE exerted dual genetic activity, clastogenic and aneugenic. Recent findings of our group have shown that nitrogen mustard analogs, melphalan (MEL), chlorambucil (CAB) and PHE, in addition to their clastogenic activity, affect chromosome segregation owing to modifications of mitotic apparatus (Efthimiou et al ., ), in contrast to the PHE steroidal analogs, EA‐92 [3b‐hydroxy‐17b‐acetamide‐androst‐5‐en‐7‐one‐4‐ N , N‐bis (2‐hloroethyl)amino phenylacetate] and EA‐97 [3b‐hydroxy‐cholest‐5‐en‐7‐one‐4‐ N , N‐bis (2‐hloroethyl)aminophenylacetate], which appeared to exclusively induce chromosome breakage in human lymphocyte cultures (Efthimiou et al ., ).…”

“…In order to investigate whether apoptosis is a mechanism for elimination of cells with chromosome damage and centrosome defects generated by MEL, CAB and PHE, apoptosis was inhibited by pan‐caspase inhibitor Z‐VAD‐FMK and immunofluorescence of β ‐ and γ ‐tubulin in the same cell system. C 2 C 12 cells, derived from mouse fibroblasts (Yaffe and Saxel, ; Blau et al ., ), have the ability to form an extensive microtubule network, making it a convenient system to study alterations in the mitotic spindle (Efthimiou et al ., ). The expression of two Aurora kinases, Aurora‐A and Aurora‐B, as well as survivin and γ ‐tubulin in cells treated with MEL, CAB and EA‐97 was studied by western blot analysis.…”