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Functional evaluation of the apoptosome in renal cell carcinoma.

Gerhard MC, Zantl N, Weirich G, Schliep S, Seiffert B, Häcker G - Br. J. Cancer (2003)

Bottom Line: Of the four nonclear cell RCC that could be included, both oncocytomas gave no response to cytochrome c (in one case, no Apaf-1 was detected), one chromophobe RCC lacked caspase-9 and failed to activate caspase-3 in response to cytochrome c, and one papillary RCC showed good caspase activation despite the lack of caspase-7.Experiments utilising a peptide derived from Smac/DIABLO gave no indication that inhibitor of apoptosis proteins might exert an inhibiting effect in primary clear cell RCC.Thus, the apoptosome signalling complex is intact in human (clear cell) RCC, and an apoptosis defect must be located at other, probably upstream, sites.

View Article: PubMed Central - PubMed

Affiliation: Institute for Medical Microbiology, Immunology and Hygiene, Trogerstrasse 9, Munich D-81675, Germany.

ABSTRACT
Renal cell carcinoma (RCC) responds very poorly to chemo- or radiotherapy. Renal cell carcinoma cell lines have been described to be resistant to apoptosis-inducing stimuli and to lack caspase expression. Here, we provide a structural and functional assessment of the apoptosome, the central caspase-activating signalling complex and a candidate for apoptosis-inactivating mutations. Cells from RCC cell lines and clinical samples isolated from RCC patients were included. Apoptosome function was measured as quantitative activation of caspases in protein extracts. In all five cell lines and in 19 out of 20 primary clear cell RCC samples, the expression of apoptosome components and caspase activation appeared normal. Of the four nonclear cell RCC that could be included, both oncocytomas gave no response to cytochrome c (in one case, no Apaf-1 was detected), one chromophobe RCC lacked caspase-9 and failed to activate caspase-3 in response to cytochrome c, and one papillary RCC showed good caspase activation despite the lack of caspase-7. Experiments utilising a peptide derived from Smac/DIABLO gave no indication that inhibitor of apoptosis proteins might exert an inhibiting effect in primary clear cell RCC. Thus, the apoptosome signalling complex is intact in human (clear cell) RCC, and an apoptosis defect must be located at other, probably upstream, sites.

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Related in: MedlinePlus

Examples of expression and processing of components of the apoptosome in clinical samples from RCC. Cells were isolated and extracts were prepared from fresh explants of clear cell RCC (A) or from one chromophobe RCC and two oncocytomas (B). Extracts from HeLa cells were prepared as above. Extracts (800 μg protein in 40 μl) were incubated for 1 h at 37°C in the presence or absence of 50 μg ml−1 cytochrome c. A measure of 200 μg per lane were run on SDS–PAGE and proteins were detected by Western blotting. Purity of the chromophobe RCC was about 80% tumour cells, suggesting that a contamination of 20% nontumour cells does not distort the results. (A) Asterisk denotes a nonspecific band, arrow procaspase-9. The several bands recognised by the anti-Apaf-1 antibody were seen in several experiments and may constitute Apaf-1 variants or, at least in part, products of nonspecific degradation. The smaller size band in the lane patient #3, no cytochrome c in the caspase-9 blot is of unknown origin and was not see in any other blot.
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fig2: Examples of expression and processing of components of the apoptosome in clinical samples from RCC. Cells were isolated and extracts were prepared from fresh explants of clear cell RCC (A) or from one chromophobe RCC and two oncocytomas (B). Extracts from HeLa cells were prepared as above. Extracts (800 μg protein in 40 μl) were incubated for 1 h at 37°C in the presence or absence of 50 μg ml−1 cytochrome c. A measure of 200 μg per lane were run on SDS–PAGE and proteins were detected by Western blotting. Purity of the chromophobe RCC was about 80% tumour cells, suggesting that a contamination of 20% nontumour cells does not distort the results. (A) Asterisk denotes a nonspecific band, arrow procaspase-9. The several bands recognised by the anti-Apaf-1 antibody were seen in several experiments and may constitute Apaf-1 variants or, at least in part, products of nonspecific degradation. The smaller size band in the lane patient #3, no cytochrome c in the caspase-9 blot is of unknown origin and was not see in any other blot.

Mentions: DEVD-cleaving activity, effect of caspase activity (see Methods).


Functional evaluation of the apoptosome in renal cell carcinoma.

Gerhard MC, Zantl N, Weirich G, Schliep S, Seiffert B, Häcker G - Br. J. Cancer (2003)

Examples of expression and processing of components of the apoptosome in clinical samples from RCC. Cells were isolated and extracts were prepared from fresh explants of clear cell RCC (A) or from one chromophobe RCC and two oncocytomas (B). Extracts from HeLa cells were prepared as above. Extracts (800 μg protein in 40 μl) were incubated for 1 h at 37°C in the presence or absence of 50 μg ml−1 cytochrome c. A measure of 200 μg per lane were run on SDS–PAGE and proteins were detected by Western blotting. Purity of the chromophobe RCC was about 80% tumour cells, suggesting that a contamination of 20% nontumour cells does not distort the results. (A) Asterisk denotes a nonspecific band, arrow procaspase-9. The several bands recognised by the anti-Apaf-1 antibody were seen in several experiments and may constitute Apaf-1 variants or, at least in part, products of nonspecific degradation. The smaller size band in the lane patient #3, no cytochrome c in the caspase-9 blot is of unknown origin and was not see in any other blot.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2376849&req=5

fig2: Examples of expression and processing of components of the apoptosome in clinical samples from RCC. Cells were isolated and extracts were prepared from fresh explants of clear cell RCC (A) or from one chromophobe RCC and two oncocytomas (B). Extracts from HeLa cells were prepared as above. Extracts (800 μg protein in 40 μl) were incubated for 1 h at 37°C in the presence or absence of 50 μg ml−1 cytochrome c. A measure of 200 μg per lane were run on SDS–PAGE and proteins were detected by Western blotting. Purity of the chromophobe RCC was about 80% tumour cells, suggesting that a contamination of 20% nontumour cells does not distort the results. (A) Asterisk denotes a nonspecific band, arrow procaspase-9. The several bands recognised by the anti-Apaf-1 antibody were seen in several experiments and may constitute Apaf-1 variants or, at least in part, products of nonspecific degradation. The smaller size band in the lane patient #3, no cytochrome c in the caspase-9 blot is of unknown origin and was not see in any other blot.
Mentions: DEVD-cleaving activity, effect of caspase activity (see Methods).

Bottom Line: Of the four nonclear cell RCC that could be included, both oncocytomas gave no response to cytochrome c (in one case, no Apaf-1 was detected), one chromophobe RCC lacked caspase-9 and failed to activate caspase-3 in response to cytochrome c, and one papillary RCC showed good caspase activation despite the lack of caspase-7.Experiments utilising a peptide derived from Smac/DIABLO gave no indication that inhibitor of apoptosis proteins might exert an inhibiting effect in primary clear cell RCC.Thus, the apoptosome signalling complex is intact in human (clear cell) RCC, and an apoptosis defect must be located at other, probably upstream, sites.

View Article: PubMed Central - PubMed

Affiliation: Institute for Medical Microbiology, Immunology and Hygiene, Trogerstrasse 9, Munich D-81675, Germany.

ABSTRACT
Renal cell carcinoma (RCC) responds very poorly to chemo- or radiotherapy. Renal cell carcinoma cell lines have been described to be resistant to apoptosis-inducing stimuli and to lack caspase expression. Here, we provide a structural and functional assessment of the apoptosome, the central caspase-activating signalling complex and a candidate for apoptosis-inactivating mutations. Cells from RCC cell lines and clinical samples isolated from RCC patients were included. Apoptosome function was measured as quantitative activation of caspases in protein extracts. In all five cell lines and in 19 out of 20 primary clear cell RCC samples, the expression of apoptosome components and caspase activation appeared normal. Of the four nonclear cell RCC that could be included, both oncocytomas gave no response to cytochrome c (in one case, no Apaf-1 was detected), one chromophobe RCC lacked caspase-9 and failed to activate caspase-3 in response to cytochrome c, and one papillary RCC showed good caspase activation despite the lack of caspase-7. Experiments utilising a peptide derived from Smac/DIABLO gave no indication that inhibitor of apoptosis proteins might exert an inhibiting effect in primary clear cell RCC. Thus, the apoptosome signalling complex is intact in human (clear cell) RCC, and an apoptosis defect must be located at other, probably upstream, sites.

Show MeSH
Related in: MedlinePlus