Overexpression of Bcl-2 is associated with apoptotic resistance to the G-quadruplex ligand 12459 but is not sufficient to confer resistance to long-term senescence

The triazine derivative 12459 is a potent G-quadruplex interacting agent that inhibits telomerase activity. This agent induces time- and dose-dependent telomere shortening, senescence-like growth arrest and apoptosis in the human A549 tumour cell line. We show here that 12459 induces a delayed apoptosis that activates the mitochondrial pathway. A549 cell lines selected for resistance to 12459 and previously characterized for an altered hTERT expression also showed Bcl-2 overexpression. Transfection of Bcl-2 into A549 cells induced a resistance to the short-term apoptotic effect triggered by 12459, suggesting that Bcl-2 is an important determinant for the activity of 12459. In sharp contrast, the Bcl-2 overexpression was not sufficient to confer resistance to the senescence-like growth arrest induced by prolonged treatment with 12459. We also show that 12459 provokes a rapid degradation of the telomeric G-overhang in conditions that paralleled the apoptosis induction. In contrast, the G-overhang degradation was not observed when apoptosis was induced by camptothecin. Bcl-2 overexpression did not modify the G-overhang degradation, suggesting that this event is an early process uncoupled from the final apoptotic pathway

Expression et fonctions de protéines impliquées dans la résistance et la sensibilté aux agents anticancéreux : nouveaux substrats des protéines ABC et nouvelles cibles de l'imatinib dans la myéloïde chronique

REIMS-BU Santé (514542104) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

( and ) Western blot analysis of PARP cleaved form (89 kDa) protein expression (a) or Caspase 3 cleaved form (17 kDa) protein expression (b) performed in untreated A549, JFD18 or JFD9 cells (0) and in cells treated with 10 μM 12459 for 24 or 48 h

<p><b>Copyright information:</b></p><p>Taken from "Overexpression of Bcl-2 is associated with apoptotic resistance to the G-quadruplex ligand 12459 but is not sufficient to confer resistance to long-term senescence"</p><p>Nucleic Acids Research 2005;33(7):2192-2203.</p><p>Published online 14 Apr 2005</p><p>PMCID:PMC1079972.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> β-actin was used as a control for protein loading. Relative PARP cleavage (a) or Caspase 3 cleavage (b) was measured by densitometry scanning of the films, normalized relative to β-actin protein expression and results were expressed relative to untreated A549 cells defined as 100% for PARP or relative to A549 cells treated for 48 h with 12459 for caspase 3 (see bottom of each panel). () Effect of caspase inhibitors on apoptosis induced by 12459 (10 μM, 48 h) on A549 cells. DEVD-FMK and IETD-FMK at 2 μM were added to 12459 and apoptosis was revealed by Hoechst 33342 staining. () Mitochondrial membrane potential was measured by spectrofluorimetry of the JC1 dye in A549 control cells (0) or in cells treated with 1 μM 12459 for 0, 6, 12 or 24 h or with 10 μM 12459 for 4 or 6 h, as described in Materials and Methods. () ROS were measured by spectrofluorimetry using carboxy fluorescein-AM in A549 control cells (0) or in cells treated with 10 μM 12459 for 2, 4, 6, 8 and 14 h. Results represent the mean ± SM of duplicate determinations

( and ) Western blot analysis of Bcl-2 and Bax protein expression performed in A549, JFD18 or JFD9 control cells (0) or cells treated with 10 μM 12459 for 24 or 48 h, as indicated

<p><b>Copyright information:</b></p><p>Taken from "Overexpression of Bcl-2 is associated with apoptotic resistance to the G-quadruplex ligand 12459 but is not sufficient to confer resistance to long-term senescence"</p><p>Nucleic Acids Research 2005;33(7):2192-2203.</p><p>Published online 14 Apr 2005</p><p>PMCID:PMC1079972.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> β-actin was used as a control for protein loading. ( and ) Analysis of Bcl-2 and Bax mRNA expression by quantitative RT–PCR in untreated A549 cells (0) and in A549 cells treated with 10 μM 12459 for 24 or 48 h. Results (mean ± SD of triplicates) were normalized relative to 18S ribosomal RNA level, and expressed relative to untreated A549 cells, defined as 100%, as described in Materials and Methods