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Cystatin C as a p53-inducible apoptotic mediator that regulates cathepsin L activity.

Mori J, Tanikawa C, Funauchi Y, Lo PH, Nakamura Y, Matsuda K - Cancer Sci. (2016)

Bottom Line: We showed that cathepsin L activity was decreased in HCT116 p53(+/+) cells after adriamycin treatment, but not in HCT116 p53(-/-) cells.We also found that knockdown of cystatin C reduced adriamycin-induced caspase-3 activation.Cystatin C expression was significantly downregulated in breast cancer cells with p53 mutations, and decreased cystatin C expression was associated with poor prognosis of breast cancer.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Medicine, Human Genome Center, Institute of Medical Science, The University of Tokyo, Minato, Tokyo, Japan.

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Cystatin C is a direct p53 target. (a) Genomic structure of the cystatin C gene. The white box indicates the location of the potential p53‐binding sequence (p53BS). Comparison of p53BS to the consensus p53‐binding sequence. R, purine; W, A or T; Y, pyrimidine. Identical nucleotides to the consensus sequence are written in capital letters. The underlined cytosine and guanine were substituted for thymine to examine the specificity of the p53‐binding site. (b) Results of luciferase assay of the genomic fragment containing p53BS with or without substitutions at p53BS. Luciferase activity is indicated relative to the activity of mock vector with SD (n = 3). Mutant p53 represents plasmid expressing p53 with a missense mutation (R175H). (c) ChIP assay was carried out using U373MG cells that were infected at an MOI of 10 with Ad‐p53 (lanes 2–4) or Ad‐LacZ (lane 1). DNA–protein complexes were immunoprecipitated with an anti‐p53 antibody (lanes 1 and 2) followed by qPCR analysis to evaluate the amount of genomic fragments containing p53‐binding sequence in p21WAF1 gene (left) and Cystatin C gene (right). Immunoprecipitates with an anti‐IgG antibody (lane 3) or in the absence of an antibody (–) (lane 4) were used as negative controls. Columns, mean; error bars, SD (n = 3).
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cas12881-fig-0003: Cystatin C is a direct p53 target. (a) Genomic structure of the cystatin C gene. The white box indicates the location of the potential p53‐binding sequence (p53BS). Comparison of p53BS to the consensus p53‐binding sequence. R, purine; W, A or T; Y, pyrimidine. Identical nucleotides to the consensus sequence are written in capital letters. The underlined cytosine and guanine were substituted for thymine to examine the specificity of the p53‐binding site. (b) Results of luciferase assay of the genomic fragment containing p53BS with or without substitutions at p53BS. Luciferase activity is indicated relative to the activity of mock vector with SD (n = 3). Mutant p53 represents plasmid expressing p53 with a missense mutation (R175H). (c) ChIP assay was carried out using U373MG cells that were infected at an MOI of 10 with Ad‐p53 (lanes 2–4) or Ad‐LacZ (lane 1). DNA–protein complexes were immunoprecipitated with an anti‐p53 antibody (lanes 1 and 2) followed by qPCR analysis to evaluate the amount of genomic fragments containing p53‐binding sequence in p21WAF1 gene (left) and Cystatin C gene (right). Immunoprecipitates with an anti‐IgG antibody (lane 3) or in the absence of an antibody (–) (lane 4) were used as negative controls. Columns, mean; error bars, SD (n = 3).

Mentions: To investigate whether cystatin C is a direct target of p53, we surveyed for the p53 binding sequence35 within the cystatin C locus and identified a potential binding site (p53BS) in the first intron (Fig. 3a). A 263‐base DNA fragment containing p53BS was amplified and subcloned upstream of the minimal promoter in pGL4.24 vector (pGL4.24/p53BS). The result of reporter assay revealed that U373MG cells transfected with pGL4.24/p53BS showed increased luciferase activity only in the presence of plasmid expressing wild‐type p53 (Fig. 3b). However, base substitutions in p53BS (pGL4.24/p53BSmut) diminished the enhancement of luciferase activity.


Cystatin C as a p53-inducible apoptotic mediator that regulates cathepsin L activity.

Mori J, Tanikawa C, Funauchi Y, Lo PH, Nakamura Y, Matsuda K - Cancer Sci. (2016)

Cystatin C is a direct p53 target. (a) Genomic structure of the cystatin C gene. The white box indicates the location of the potential p53‐binding sequence (p53BS). Comparison of p53BS to the consensus p53‐binding sequence. R, purine; W, A or T; Y, pyrimidine. Identical nucleotides to the consensus sequence are written in capital letters. The underlined cytosine and guanine were substituted for thymine to examine the specificity of the p53‐binding site. (b) Results of luciferase assay of the genomic fragment containing p53BS with or without substitutions at p53BS. Luciferase activity is indicated relative to the activity of mock vector with SD (n = 3). Mutant p53 represents plasmid expressing p53 with a missense mutation (R175H). (c) ChIP assay was carried out using U373MG cells that were infected at an MOI of 10 with Ad‐p53 (lanes 2–4) or Ad‐LacZ (lane 1). DNA–protein complexes were immunoprecipitated with an anti‐p53 antibody (lanes 1 and 2) followed by qPCR analysis to evaluate the amount of genomic fragments containing p53‐binding sequence in p21WAF1 gene (left) and Cystatin C gene (right). Immunoprecipitates with an anti‐IgG antibody (lane 3) or in the absence of an antibody (–) (lane 4) were used as negative controls. Columns, mean; error bars, SD (n = 3).
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cas12881-fig-0003: Cystatin C is a direct p53 target. (a) Genomic structure of the cystatin C gene. The white box indicates the location of the potential p53‐binding sequence (p53BS). Comparison of p53BS to the consensus p53‐binding sequence. R, purine; W, A or T; Y, pyrimidine. Identical nucleotides to the consensus sequence are written in capital letters. The underlined cytosine and guanine were substituted for thymine to examine the specificity of the p53‐binding site. (b) Results of luciferase assay of the genomic fragment containing p53BS with or without substitutions at p53BS. Luciferase activity is indicated relative to the activity of mock vector with SD (n = 3). Mutant p53 represents plasmid expressing p53 with a missense mutation (R175H). (c) ChIP assay was carried out using U373MG cells that were infected at an MOI of 10 with Ad‐p53 (lanes 2–4) or Ad‐LacZ (lane 1). DNA–protein complexes were immunoprecipitated with an anti‐p53 antibody (lanes 1 and 2) followed by qPCR analysis to evaluate the amount of genomic fragments containing p53‐binding sequence in p21WAF1 gene (left) and Cystatin C gene (right). Immunoprecipitates with an anti‐IgG antibody (lane 3) or in the absence of an antibody (–) (lane 4) were used as negative controls. Columns, mean; error bars, SD (n = 3).
Mentions: To investigate whether cystatin C is a direct target of p53, we surveyed for the p53 binding sequence35 within the cystatin C locus and identified a potential binding site (p53BS) in the first intron (Fig. 3a). A 263‐base DNA fragment containing p53BS was amplified and subcloned upstream of the minimal promoter in pGL4.24 vector (pGL4.24/p53BS). The result of reporter assay revealed that U373MG cells transfected with pGL4.24/p53BS showed increased luciferase activity only in the presence of plasmid expressing wild‐type p53 (Fig. 3b). However, base substitutions in p53BS (pGL4.24/p53BSmut) diminished the enhancement of luciferase activity.

Bottom Line: We showed that cathepsin L activity was decreased in HCT116 p53(+/+) cells after adriamycin treatment, but not in HCT116 p53(-/-) cells.We also found that knockdown of cystatin C reduced adriamycin-induced caspase-3 activation.Cystatin C expression was significantly downregulated in breast cancer cells with p53 mutations, and decreased cystatin C expression was associated with poor prognosis of breast cancer.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Medicine, Human Genome Center, Institute of Medical Science, The University of Tokyo, Minato, Tokyo, Japan.

Show MeSH
Related in: MedlinePlus