Limits...
Anisomycin activates JNK and sensitises DU 145 prostate carcinoma cells to Fas mediated apoptosis.

Curtin JF, Cotter TG - Br. J. Cancer (2002)

Bottom Line: Our group has shown that inhibition of JNK activity completely abrogates the effects of chemotherapeutic drugs.Inhibition of Caspase 8 and Caspase 9 completely inhibits this process which suggests that DU 145 cells require mitochondrial amplification of the Fas apoptotic signal.Furthermore, we have shown that inhibition of Fas mediated apoptosis is an early event in DU 145 cells, occurring upstream of Caspase 8 cleavage.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University College Cork, Lee Maltings, Prospect Row, Cork, Ireland.

Show MeSH

Related in: MedlinePlus

Inhibition of Fas mediated apoptosis occurs upstream of Caspase 8 cleavage in DU 145 cells. (A) Western blot analysis of Caspase 8 in untreated DU 145 cells (1) or following incubation with anisomycin (250 ng ml−1) (2), anti-Fas IgM (200 ng ml−1) (3) or both (4) for 8 h. Untreated (5) and anti-Fas IgM treated (200 ng ml−1 anti-Fas IgM, 4 h) (6) Jurkat cells were used as a positive control for the P14 and P10 Caspase 8 cleavage products. β-Actin was also probed to ensure equal protein loading. (B) Bid expression and cleavage was analysed by Western blot in untreated DU 145 cells (1) or in cells incubated with anisomycin (250 ng ml−1) (2), anti-Fas IgM (200 ng ml−1) (3) or both (4) for 8 h. Jurkats untreated (5) or treated with anti-Fas IgM (200 ng ml−1) for 4 h (6) are used as a positive control. (C) DU 145 cells were pre-treated with 50 μM z-IETD-fmk (white columns) or a DMSO control (black columns) for 10 min before treating with anisomycin and anti-Fas IgM as before. Apoptosis was determined by staining with both Annexin V and PI after 8 h. Data is representative of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2376200&req=5

fig4: Inhibition of Fas mediated apoptosis occurs upstream of Caspase 8 cleavage in DU 145 cells. (A) Western blot analysis of Caspase 8 in untreated DU 145 cells (1) or following incubation with anisomycin (250 ng ml−1) (2), anti-Fas IgM (200 ng ml−1) (3) or both (4) for 8 h. Untreated (5) and anti-Fas IgM treated (200 ng ml−1 anti-Fas IgM, 4 h) (6) Jurkat cells were used as a positive control for the P14 and P10 Caspase 8 cleavage products. β-Actin was also probed to ensure equal protein loading. (B) Bid expression and cleavage was analysed by Western blot in untreated DU 145 cells (1) or in cells incubated with anisomycin (250 ng ml−1) (2), anti-Fas IgM (200 ng ml−1) (3) or both (4) for 8 h. Jurkats untreated (5) or treated with anti-Fas IgM (200 ng ml−1) for 4 h (6) are used as a positive control. (C) DU 145 cells were pre-treated with 50 μM z-IETD-fmk (white columns) or a DMSO control (black columns) for 10 min before treating with anisomycin and anti-Fas IgM as before. Apoptosis was determined by staining with both Annexin V and PI after 8 h. Data is representative of three independent experiments.

Mentions: The proximal caspase in the Fas apoptotic pathway is Caspase 8. Recruitment and auto-cleavage of Procaspase 8 occurs following Fas receptor activation in sensitive cells. Active Caspase 8 is tetrameric, consisting of two P14 and two P10 subunits. Using Western blot analysis, we found that Caspase 8 is not cleaved in DU 145 cells following treatment with anti-Fas IgM (200 ng ml−1) (Figure 4AFigure 4


Anisomycin activates JNK and sensitises DU 145 prostate carcinoma cells to Fas mediated apoptosis.

Curtin JF, Cotter TG - Br. J. Cancer (2002)

Inhibition of Fas mediated apoptosis occurs upstream of Caspase 8 cleavage in DU 145 cells. (A) Western blot analysis of Caspase 8 in untreated DU 145 cells (1) or following incubation with anisomycin (250 ng ml−1) (2), anti-Fas IgM (200 ng ml−1) (3) or both (4) for 8 h. Untreated (5) and anti-Fas IgM treated (200 ng ml−1 anti-Fas IgM, 4 h) (6) Jurkat cells were used as a positive control for the P14 and P10 Caspase 8 cleavage products. β-Actin was also probed to ensure equal protein loading. (B) Bid expression and cleavage was analysed by Western blot in untreated DU 145 cells (1) or in cells incubated with anisomycin (250 ng ml−1) (2), anti-Fas IgM (200 ng ml−1) (3) or both (4) for 8 h. Jurkats untreated (5) or treated with anti-Fas IgM (200 ng ml−1) for 4 h (6) are used as a positive control. (C) DU 145 cells were pre-treated with 50 μM z-IETD-fmk (white columns) or a DMSO control (black columns) for 10 min before treating with anisomycin and anti-Fas IgM as before. Apoptosis was determined by staining with both Annexin V and PI after 8 h. Data is representative of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2376200&req=5

fig4: Inhibition of Fas mediated apoptosis occurs upstream of Caspase 8 cleavage in DU 145 cells. (A) Western blot analysis of Caspase 8 in untreated DU 145 cells (1) or following incubation with anisomycin (250 ng ml−1) (2), anti-Fas IgM (200 ng ml−1) (3) or both (4) for 8 h. Untreated (5) and anti-Fas IgM treated (200 ng ml−1 anti-Fas IgM, 4 h) (6) Jurkat cells were used as a positive control for the P14 and P10 Caspase 8 cleavage products. β-Actin was also probed to ensure equal protein loading. (B) Bid expression and cleavage was analysed by Western blot in untreated DU 145 cells (1) or in cells incubated with anisomycin (250 ng ml−1) (2), anti-Fas IgM (200 ng ml−1) (3) or both (4) for 8 h. Jurkats untreated (5) or treated with anti-Fas IgM (200 ng ml−1) for 4 h (6) are used as a positive control. (C) DU 145 cells were pre-treated with 50 μM z-IETD-fmk (white columns) or a DMSO control (black columns) for 10 min before treating with anisomycin and anti-Fas IgM as before. Apoptosis was determined by staining with both Annexin V and PI after 8 h. Data is representative of three independent experiments.
Mentions: The proximal caspase in the Fas apoptotic pathway is Caspase 8. Recruitment and auto-cleavage of Procaspase 8 occurs following Fas receptor activation in sensitive cells. Active Caspase 8 is tetrameric, consisting of two P14 and two P10 subunits. Using Western blot analysis, we found that Caspase 8 is not cleaved in DU 145 cells following treatment with anti-Fas IgM (200 ng ml−1) (Figure 4AFigure 4

Bottom Line: Our group has shown that inhibition of JNK activity completely abrogates the effects of chemotherapeutic drugs.Inhibition of Caspase 8 and Caspase 9 completely inhibits this process which suggests that DU 145 cells require mitochondrial amplification of the Fas apoptotic signal.Furthermore, we have shown that inhibition of Fas mediated apoptosis is an early event in DU 145 cells, occurring upstream of Caspase 8 cleavage.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University College Cork, Lee Maltings, Prospect Row, Cork, Ireland.

Show MeSH
Related in: MedlinePlus