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Induction of DNA breaks and apoptosis in crosslink-hypersensitive V79 cells by the cytostatic drug beta-D-glucosyl-ifosfamide mustard.

Becker R, Ritter A, Eichhorn U, Lips J, Bertram B, Wiessler M, Zdzienicka MZ, Kaina B - Br. J. Cancer (2002)

Bottom Line: The resistant parental cells were refractory to all these parameters.Bcl-2 decline in the sensitive cells preceded apoptosis, and transfection-mediated overexpression of Bcl-2 protected at least in part from apoptosis.From the data we hypothesize that non-repaired crosslinks induced by beta-D-glucose-ifosfamide mustard are transformed into double-strand breaks which trigger apoptosis via a Bcl-2 dependent pathway.

View Article: PubMed Central - PubMed

Affiliation: Institute of Toxicology, Division of Applied Toxicology, University of Mainz, Obere Zahlbacher Str. 67, D-55131 Mainz, Germany. kaina@mail.uni-mainz.de

ABSTRACT
To study molecular aspects of cytotoxicity of the anticancer drug beta-D-glucose-ifosfamide mustard we investigated the potential of the agent to induce apoptosis and DNA breakage. Since beta-D-glucose-ifosfamide mustard generates DNA interstrand crosslinks, we used as an in vitro model system a pair of isogenic Chinese hamster V79 cells differing in their sensitivity to crosslinking agents. CL-V5B cells are dramatically more sensitive (30-fold based on D(10) values) to the cytotoxic effects of beta-D-glucose-ifosfamide mustard as compared to parental V79B cells. After 48 h of pulse-treatment with the agent, sensitive cells but not the resistant parental line undergo apoptosis and necrosis, with apoptosis being the predominant form of cell death (70 and 20% of apoptosis and necrosis, respectively). Apoptosis increased as a function of dose and was accompanied by induction of DNA double-strand breaks in the hypersensitive cells. Furthermore, a strong decline in the level of Bcl-2 protein and activation of caspases-3, -8 and -9 were observed. The resistant parental cells were refractory to all these parameters. Bcl-2 decline in the sensitive cells preceded apoptosis, and transfection-mediated overexpression of Bcl-2 protected at least in part from apoptosis. From the data we hypothesize that non-repaired crosslinks induced by beta-D-glucose-ifosfamide mustard are transformed into double-strand breaks which trigger apoptosis via a Bcl-2 dependent pathway.

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Activation of caspase-3 (A), caspase-8 (B) and caspase-9 (C) as a function of dose. Cells were pulse-treated with β-D-Glc-IPM for 1 h. Capase activity was measured 72 h after treatment. Activities are shown as x-fold increase over control levels. Given are mean values from three experiments ±s.d. •, V79B; ▪, CL-V5B.
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fig8: Activation of caspase-3 (A), caspase-8 (B) and caspase-9 (C) as a function of dose. Cells were pulse-treated with β-D-Glc-IPM for 1 h. Capase activity was measured 72 h after treatment. Activities are shown as x-fold increase over control levels. Given are mean values from three experiments ±s.d. •, V79B; ▪, CL-V5B.

Mentions: To further elucidate the molecular mechanism of apoptosis, we investigated the activation of caspases-3, -8 and -9, which are the major executioners of drug-induced apoptosis in many cell systems. As shown in Figure 8Figure 8


Induction of DNA breaks and apoptosis in crosslink-hypersensitive V79 cells by the cytostatic drug beta-D-glucosyl-ifosfamide mustard.

Becker R, Ritter A, Eichhorn U, Lips J, Bertram B, Wiessler M, Zdzienicka MZ, Kaina B - Br. J. Cancer (2002)

Activation of caspase-3 (A), caspase-8 (B) and caspase-9 (C) as a function of dose. Cells were pulse-treated with β-D-Glc-IPM for 1 h. Capase activity was measured 72 h after treatment. Activities are shown as x-fold increase over control levels. Given are mean values from three experiments ±s.d. •, V79B; ▪, CL-V5B.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2746532&req=5

fig8: Activation of caspase-3 (A), caspase-8 (B) and caspase-9 (C) as a function of dose. Cells were pulse-treated with β-D-Glc-IPM for 1 h. Capase activity was measured 72 h after treatment. Activities are shown as x-fold increase over control levels. Given are mean values from three experiments ±s.d. •, V79B; ▪, CL-V5B.
Mentions: To further elucidate the molecular mechanism of apoptosis, we investigated the activation of caspases-3, -8 and -9, which are the major executioners of drug-induced apoptosis in many cell systems. As shown in Figure 8Figure 8

Bottom Line: The resistant parental cells were refractory to all these parameters.Bcl-2 decline in the sensitive cells preceded apoptosis, and transfection-mediated overexpression of Bcl-2 protected at least in part from apoptosis.From the data we hypothesize that non-repaired crosslinks induced by beta-D-glucose-ifosfamide mustard are transformed into double-strand breaks which trigger apoptosis via a Bcl-2 dependent pathway.

View Article: PubMed Central - PubMed

Affiliation: Institute of Toxicology, Division of Applied Toxicology, University of Mainz, Obere Zahlbacher Str. 67, D-55131 Mainz, Germany. kaina@mail.uni-mainz.de

ABSTRACT
To study molecular aspects of cytotoxicity of the anticancer drug beta-D-glucose-ifosfamide mustard we investigated the potential of the agent to induce apoptosis and DNA breakage. Since beta-D-glucose-ifosfamide mustard generates DNA interstrand crosslinks, we used as an in vitro model system a pair of isogenic Chinese hamster V79 cells differing in their sensitivity to crosslinking agents. CL-V5B cells are dramatically more sensitive (30-fold based on D(10) values) to the cytotoxic effects of beta-D-glucose-ifosfamide mustard as compared to parental V79B cells. After 48 h of pulse-treatment with the agent, sensitive cells but not the resistant parental line undergo apoptosis and necrosis, with apoptosis being the predominant form of cell death (70 and 20% of apoptosis and necrosis, respectively). Apoptosis increased as a function of dose and was accompanied by induction of DNA double-strand breaks in the hypersensitive cells. Furthermore, a strong decline in the level of Bcl-2 protein and activation of caspases-3, -8 and -9 were observed. The resistant parental cells were refractory to all these parameters. Bcl-2 decline in the sensitive cells preceded apoptosis, and transfection-mediated overexpression of Bcl-2 protected at least in part from apoptosis. From the data we hypothesize that non-repaired crosslinks induced by beta-D-glucose-ifosfamide mustard are transformed into double-strand breaks which trigger apoptosis via a Bcl-2 dependent pathway.

Show MeSH
Related in: MedlinePlus