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Expression of TP53 isoforms p53β or p53γ enhances chemosensitivity in TP53() cell lines.

Silden E, Hjelle SM, Wergeland L, Sulen A, Andresen V, Bourdon JC, Micklem DR, McCormack E, Gjertsen BT - PLoS ONE (2013)

Bottom Line: Treatment with dicoumarol, a putative blocker of the proteasome-related NAD(P)H quinone oxidoreductase NQO1, effectively attenuated basal p53γ protein level in spite of bortezomib treatment.Although in vitro proliferation and clonogenicity assays indicated a weak suppressive effect by p53β and p53γ expression, studies of in vivo subcutaneous H1299 tumor growth demonstrated a significantly increased growth by expression of either p53 isoforms.This study suggests that p53β and p53γ share functionality in chemosensitizing and tumor growth enhancement but comprise distinct regulation at the protein level.

View Article: PubMed Central - PubMed

Affiliation: Hematology Section, Institute of Medicine, University of Bergen, Bergen, Norway.

ABSTRACT
The carboxy-terminal truncated p53 alternative spliced isoforms, p53β and p53γ, are expressed at disparate levels in cancer and are suggested to influence treatment response and therapy outcome. However, their functional role in cancer remains to be elucidated. We investigated their individual functionality in the p53() background of cell lines H1299 and SAOS-2 by stable retroviral transduction or transient transfection. Expression status of p53β and p53γ protein was found to correlate with increased response to camptothecin and doxorubicin chemotherapy. Decreased DNA synthesis and clonogenicity in p53β and p53γ congenic H1299 was accompanied by increased p21((CIP1/WAF1)), Bax and Mdm2 proteins. Chemotherapy induced p53 isoform degradation, most prominent for p53γ. The proteasome inhibitor bortezomib substantially increased basal p53γ protein level, while the level of p53β protein was unaffected. Treatment with dicoumarol, a putative blocker of the proteasome-related NAD(P)H quinone oxidoreductase NQO1, effectively attenuated basal p53γ protein level in spite of bortezomib treatment. Although in vitro proliferation and clonogenicity assays indicated a weak suppressive effect by p53β and p53γ expression, studies of in vivo subcutaneous H1299 tumor growth demonstrated a significantly increased growth by expression of either p53 isoforms. This study suggests that p53β and p53γ share functionality in chemosensitizing and tumor growth enhancement but comprise distinct regulation at the protein level.

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p53 isoforms and experimental setup.(A) The human TP53 gene have alternative promoters (P1, P1′, P2) and several alternative splicing sites (full-length p53 (α), β, γ) generating p53 isoforms. Alternative splicing of intron 9 leads to expression of the p53 protein isoforms p53β and p53γ with a truncated carboxy-terminal terminating with 10 and 15 additional amino acids, respectively. Binding site of p53 antibodies Bp53-12 (recognizes amino-acid region 16–25), DO-1 (amino-acids 20–25) and DO-7 (amino-acids 37–45) is indicated. NLS: nuclear localization signal. TD: tetramerization domain. BD: basic domain. (B) p53 H1299 lung carcinoma cell line were retrovirally transduced with plasmid vector containing p53 isoforms p53β, p53γ or p53FL and a tdTomato reporter. TdTomato expression allows FACS sorting of successfully transduced cells. (C) Fluorescence microscopy confirms tdTomato expression (red) of FACS sorted H1299 cells. Scale bar: 100 µm. p53 (DO-1) immunofluorescence staining (green) show mainly nuclear localization of p53β and both nuclear and cytoplasmic localization of p53γ. DAPI (blue) DNA stain visualize the nucleus. Scale bar: 20 µm.
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pone-0056276-g001: p53 isoforms and experimental setup.(A) The human TP53 gene have alternative promoters (P1, P1′, P2) and several alternative splicing sites (full-length p53 (α), β, γ) generating p53 isoforms. Alternative splicing of intron 9 leads to expression of the p53 protein isoforms p53β and p53γ with a truncated carboxy-terminal terminating with 10 and 15 additional amino acids, respectively. Binding site of p53 antibodies Bp53-12 (recognizes amino-acid region 16–25), DO-1 (amino-acids 20–25) and DO-7 (amino-acids 37–45) is indicated. NLS: nuclear localization signal. TD: tetramerization domain. BD: basic domain. (B) p53 H1299 lung carcinoma cell line were retrovirally transduced with plasmid vector containing p53 isoforms p53β, p53γ or p53FL and a tdTomato reporter. TdTomato expression allows FACS sorting of successfully transduced cells. (C) Fluorescence microscopy confirms tdTomato expression (red) of FACS sorted H1299 cells. Scale bar: 100 µm. p53 (DO-1) immunofluorescence staining (green) show mainly nuclear localization of p53β and both nuclear and cytoplasmic localization of p53γ. DAPI (blue) DNA stain visualize the nucleus. Scale bar: 20 µm.

Mentions: The gene of the tumor suppressor p53 is shown to encode at least 12 different p53 protein isoforms through alternative splicing, promoter and translational initiation (reviewed in [1], [2]) (Figure 1A). The differential expression of several of these isoforms has recently been established in cancer, [3], [4] though their functional role is not fully understood. Their structural characteristics may indicate isoform specific mechanisms. p53β and p53γ lack the oligomerization domain (Figure 1A) that is required for p53 tetramerization and thus influence p53 DNA binding and transcriptional activity. However, p53β has been shown to bind certain p53 promoters and form protein complexes with full-length p53. Furthermore, p53β and p53γ is expressed in a tissue-specific manner, which may suggest diverse tissue-determined functions that may be reflected in cancer [5]. This complicates a simple understanding of p53 function, but may support future use of p53 isoform profiles in prediction of outcome and drug sensitivity in cancers.


Expression of TP53 isoforms p53β or p53γ enhances chemosensitivity in TP53() cell lines.

Silden E, Hjelle SM, Wergeland L, Sulen A, Andresen V, Bourdon JC, Micklem DR, McCormack E, Gjertsen BT - PLoS ONE (2013)

p53 isoforms and experimental setup.(A) The human TP53 gene have alternative promoters (P1, P1′, P2) and several alternative splicing sites (full-length p53 (α), β, γ) generating p53 isoforms. Alternative splicing of intron 9 leads to expression of the p53 protein isoforms p53β and p53γ with a truncated carboxy-terminal terminating with 10 and 15 additional amino acids, respectively. Binding site of p53 antibodies Bp53-12 (recognizes amino-acid region 16–25), DO-1 (amino-acids 20–25) and DO-7 (amino-acids 37–45) is indicated. NLS: nuclear localization signal. TD: tetramerization domain. BD: basic domain. (B) p53 H1299 lung carcinoma cell line were retrovirally transduced with plasmid vector containing p53 isoforms p53β, p53γ or p53FL and a tdTomato reporter. TdTomato expression allows FACS sorting of successfully transduced cells. (C) Fluorescence microscopy confirms tdTomato expression (red) of FACS sorted H1299 cells. Scale bar: 100 µm. p53 (DO-1) immunofluorescence staining (green) show mainly nuclear localization of p53β and both nuclear and cytoplasmic localization of p53γ. DAPI (blue) DNA stain visualize the nucleus. Scale bar: 20 µm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3569410&req=5

pone-0056276-g001: p53 isoforms and experimental setup.(A) The human TP53 gene have alternative promoters (P1, P1′, P2) and several alternative splicing sites (full-length p53 (α), β, γ) generating p53 isoforms. Alternative splicing of intron 9 leads to expression of the p53 protein isoforms p53β and p53γ with a truncated carboxy-terminal terminating with 10 and 15 additional amino acids, respectively. Binding site of p53 antibodies Bp53-12 (recognizes amino-acid region 16–25), DO-1 (amino-acids 20–25) and DO-7 (amino-acids 37–45) is indicated. NLS: nuclear localization signal. TD: tetramerization domain. BD: basic domain. (B) p53 H1299 lung carcinoma cell line were retrovirally transduced with plasmid vector containing p53 isoforms p53β, p53γ or p53FL and a tdTomato reporter. TdTomato expression allows FACS sorting of successfully transduced cells. (C) Fluorescence microscopy confirms tdTomato expression (red) of FACS sorted H1299 cells. Scale bar: 100 µm. p53 (DO-1) immunofluorescence staining (green) show mainly nuclear localization of p53β and both nuclear and cytoplasmic localization of p53γ. DAPI (blue) DNA stain visualize the nucleus. Scale bar: 20 µm.
Mentions: The gene of the tumor suppressor p53 is shown to encode at least 12 different p53 protein isoforms through alternative splicing, promoter and translational initiation (reviewed in [1], [2]) (Figure 1A). The differential expression of several of these isoforms has recently been established in cancer, [3], [4] though their functional role is not fully understood. Their structural characteristics may indicate isoform specific mechanisms. p53β and p53γ lack the oligomerization domain (Figure 1A) that is required for p53 tetramerization and thus influence p53 DNA binding and transcriptional activity. However, p53β has been shown to bind certain p53 promoters and form protein complexes with full-length p53. Furthermore, p53β and p53γ is expressed in a tissue-specific manner, which may suggest diverse tissue-determined functions that may be reflected in cancer [5]. This complicates a simple understanding of p53 function, but may support future use of p53 isoform profiles in prediction of outcome and drug sensitivity in cancers.

Bottom Line: Treatment with dicoumarol, a putative blocker of the proteasome-related NAD(P)H quinone oxidoreductase NQO1, effectively attenuated basal p53γ protein level in spite of bortezomib treatment.Although in vitro proliferation and clonogenicity assays indicated a weak suppressive effect by p53β and p53γ expression, studies of in vivo subcutaneous H1299 tumor growth demonstrated a significantly increased growth by expression of either p53 isoforms.This study suggests that p53β and p53γ share functionality in chemosensitizing and tumor growth enhancement but comprise distinct regulation at the protein level.

View Article: PubMed Central - PubMed

Affiliation: Hematology Section, Institute of Medicine, University of Bergen, Bergen, Norway.

ABSTRACT
The carboxy-terminal truncated p53 alternative spliced isoforms, p53β and p53γ, are expressed at disparate levels in cancer and are suggested to influence treatment response and therapy outcome. However, their functional role in cancer remains to be elucidated. We investigated their individual functionality in the p53() background of cell lines H1299 and SAOS-2 by stable retroviral transduction or transient transfection. Expression status of p53β and p53γ protein was found to correlate with increased response to camptothecin and doxorubicin chemotherapy. Decreased DNA synthesis and clonogenicity in p53β and p53γ congenic H1299 was accompanied by increased p21((CIP1/WAF1)), Bax and Mdm2 proteins. Chemotherapy induced p53 isoform degradation, most prominent for p53γ. The proteasome inhibitor bortezomib substantially increased basal p53γ protein level, while the level of p53β protein was unaffected. Treatment with dicoumarol, a putative blocker of the proteasome-related NAD(P)H quinone oxidoreductase NQO1, effectively attenuated basal p53γ protein level in spite of bortezomib treatment. Although in vitro proliferation and clonogenicity assays indicated a weak suppressive effect by p53β and p53γ expression, studies of in vivo subcutaneous H1299 tumor growth demonstrated a significantly increased growth by expression of either p53 isoforms. This study suggests that p53β and p53γ share functionality in chemosensitizing and tumor growth enhancement but comprise distinct regulation at the protein level.

Show MeSH
Related in: MedlinePlus