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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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Protein stabilization of p53β and p53γ.(A) Immunoblot of p53γ and p53β protein after treatment with 50 nM proteasome inhibitor bortezomib (Bzm) or 100 µM lysosome inhibitor chloroquine (Chq) for 8 hrs. (B) Immunofluorescence images of H1299 p53γ and H1299 p53β−/+bortezomib treatment (Bzm; 50 nM, 8 hrs). p53 (DO-1) immunofluorescence staining in green. DAPI (blue): nucleic acid stain. i) shows the differences in expression levels (10× magnification), while ii) enlightens the subcellular localization (63× magnification). (C) Treatment of H1299 p53γ (top) and H1299 p53β (bottom) with doxorubicin (0.5 µM) and nutlin-3 (10 µM) for 8 hrs. GAPDH is included as a loading control. The ratio of p53 to loading control compared to the untreated vector control cells (set to 1.0) is indicated. (D) Treatment of H1299 p53γ (left) and H1299 p53β (right) with NQO1-inhibitor dicoumarol for 8 hrs. Quantification of protein signal presented as ratio of p53 to β-actin in lower panel (n = 2 immunoblots). β-actin was included as a loading control. Error bars: standard error of mean. (E) Protein levels of p53γ and Mdm2 after treatment of H1299 p53γ with 0.5 µM doxorubicin (Dox), 200 µM dicoumarol (Dic) and 50 nM bortezomib (Bzm) for 8 hours (a representative immunoblot shown). GAPDH is included as a loading control.
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pone-0056276-g005: Protein stabilization of p53β and p53γ.(A) Immunoblot of p53γ and p53β protein after treatment with 50 nM proteasome inhibitor bortezomib (Bzm) or 100 µM lysosome inhibitor chloroquine (Chq) for 8 hrs. (B) Immunofluorescence images of H1299 p53γ and H1299 p53β−/+bortezomib treatment (Bzm; 50 nM, 8 hrs). p53 (DO-1) immunofluorescence staining in green. DAPI (blue): nucleic acid stain. i) shows the differences in expression levels (10× magnification), while ii) enlightens the subcellular localization (63× magnification). (C) Treatment of H1299 p53γ (top) and H1299 p53β (bottom) with doxorubicin (0.5 µM) and nutlin-3 (10 µM) for 8 hrs. GAPDH is included as a loading control. The ratio of p53 to loading control compared to the untreated vector control cells (set to 1.0) is indicated. (D) Treatment of H1299 p53γ (left) and H1299 p53β (right) with NQO1-inhibitor dicoumarol for 8 hrs. Quantification of protein signal presented as ratio of p53 to β-actin in lower panel (n = 2 immunoblots). β-actin was included as a loading control. Error bars: standard error of mean. (E) Protein levels of p53γ and Mdm2 after treatment of H1299 p53γ with 0.5 µM doxorubicin (Dox), 200 µM dicoumarol (Dic) and 50 nM bortezomib (Bzm) for 8 hours (a representative immunoblot shown). GAPDH is included as a loading control.

Mentions: The p53γ protein was considerably degraded after doxorubicin exposure (Figure 4A). To investigate the mechanism of degradation, H1299 p53β and H1299 p53γ cells were treated with either the proteasome inhibitor bortezomib (Bzm) or the lysosome inhibitor chloroquine (Chq). Immunoblotting demonstrated that while p53γ levels were elevated considerably subsequent to treatment with bortezomib, p53β displayed stable protein levels after bortezomib and chloroquine treatment (Figure 5A). These findings was further confirmed at the subcellular level through immunofluorescence of the H1299 p53β and H1299 p53γ cells (Figure 5B) where an increase in fluorescence was observed for p53γ after 8 hours of bortezomib treatment while the level of p53β appeared unaffected (Figure 5B (i)). An incubation time of 8 hours with bortezomib was needed in order to inhibit p53γ degradation since treatment for 1 hour, 2 hours and 4.5 hours did not result in an inhibition of p53γ degradation and also no difference in the expression level of p53β was observed using these time points (data not shown). To further investigate the subcellular localization of p53γ and p53β after bortezomib treatment as compared to untreated cells, the cells was investigated using a higher magnification where the untreated p53γ cells were overexposed in order to capture the localization (Figure 5B ii)). It is evident in both untreated and bortezomib treated cells that p53γ is concentrated in the nucleus, excluded from the nucleoli and only observed diffusely in the cytoplasm. Without treatment, the p53β protein was concentrated in the nucleus, localized in the nucleoli and in a speckled nucleoplasmic pattern, in addition to diffusely in the cytoplasm. After bortezomib treatment the nuclear localization of p53β appeared more diffuse. No change in p53γ and p53β stability or subcellular localization was observed after exposure to chloroquine at the time points described above (data not shown).


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)

Protein stabilization of p53β and p53γ.(A) Immunoblot of p53γ and p53β protein after treatment with 50 nM proteasome inhibitor bortezomib (Bzm) or 100 µM lysosome inhibitor chloroquine (Chq) for 8 hrs. (B) Immunofluorescence images of H1299 p53γ and H1299 p53β−/+bortezomib treatment (Bzm; 50 nM, 8 hrs). p53 (DO-1) immunofluorescence staining in green. DAPI (blue): nucleic acid stain. i) shows the differences in expression levels (10× magnification), while ii) enlightens the subcellular localization (63× magnification). (C) Treatment of H1299 p53γ (top) and H1299 p53β (bottom) with doxorubicin (0.5 µM) and nutlin-3 (10 µM) for 8 hrs. GAPDH is included as a loading control. The ratio of p53 to loading control compared to the untreated vector control cells (set to 1.0) is indicated. (D) Treatment of H1299 p53γ (left) and H1299 p53β (right) with NQO1-inhibitor dicoumarol for 8 hrs. Quantification of protein signal presented as ratio of p53 to β-actin in lower panel (n = 2 immunoblots). β-actin was included as a loading control. Error bars: standard error of mean. (E) Protein levels of p53γ and Mdm2 after treatment of H1299 p53γ with 0.5 µM doxorubicin (Dox), 200 µM dicoumarol (Dic) and 50 nM bortezomib (Bzm) for 8 hours (a representative immunoblot shown). GAPDH is included as a loading control.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3569410&req=5

pone-0056276-g005: Protein stabilization of p53β and p53γ.(A) Immunoblot of p53γ and p53β protein after treatment with 50 nM proteasome inhibitor bortezomib (Bzm) or 100 µM lysosome inhibitor chloroquine (Chq) for 8 hrs. (B) Immunofluorescence images of H1299 p53γ and H1299 p53β−/+bortezomib treatment (Bzm; 50 nM, 8 hrs). p53 (DO-1) immunofluorescence staining in green. DAPI (blue): nucleic acid stain. i) shows the differences in expression levels (10× magnification), while ii) enlightens the subcellular localization (63× magnification). (C) Treatment of H1299 p53γ (top) and H1299 p53β (bottom) with doxorubicin (0.5 µM) and nutlin-3 (10 µM) for 8 hrs. GAPDH is included as a loading control. The ratio of p53 to loading control compared to the untreated vector control cells (set to 1.0) is indicated. (D) Treatment of H1299 p53γ (left) and H1299 p53β (right) with NQO1-inhibitor dicoumarol for 8 hrs. Quantification of protein signal presented as ratio of p53 to β-actin in lower panel (n = 2 immunoblots). β-actin was included as a loading control. Error bars: standard error of mean. (E) Protein levels of p53γ and Mdm2 after treatment of H1299 p53γ with 0.5 µM doxorubicin (Dox), 200 µM dicoumarol (Dic) and 50 nM bortezomib (Bzm) for 8 hours (a representative immunoblot shown). GAPDH is included as a loading control.
Mentions: The p53γ protein was considerably degraded after doxorubicin exposure (Figure 4A). To investigate the mechanism of degradation, H1299 p53β and H1299 p53γ cells were treated with either the proteasome inhibitor bortezomib (Bzm) or the lysosome inhibitor chloroquine (Chq). Immunoblotting demonstrated that while p53γ levels were elevated considerably subsequent to treatment with bortezomib, p53β displayed stable protein levels after bortezomib and chloroquine treatment (Figure 5A). These findings was further confirmed at the subcellular level through immunofluorescence of the H1299 p53β and H1299 p53γ cells (Figure 5B) where an increase in fluorescence was observed for p53γ after 8 hours of bortezomib treatment while the level of p53β appeared unaffected (Figure 5B (i)). An incubation time of 8 hours with bortezomib was needed in order to inhibit p53γ degradation since treatment for 1 hour, 2 hours and 4.5 hours did not result in an inhibition of p53γ degradation and also no difference in the expression level of p53β was observed using these time points (data not shown). To further investigate the subcellular localization of p53γ and p53β after bortezomib treatment as compared to untreated cells, the cells was investigated using a higher magnification where the untreated p53γ cells were overexposed in order to capture the localization (Figure 5B ii)). It is evident in both untreated and bortezomib treated cells that p53γ is concentrated in the nucleus, excluded from the nucleoli and only observed diffusely in the cytoplasm. Without treatment, the p53β protein was concentrated in the nucleus, localized in the nucleoli and in a speckled nucleoplasmic pattern, in addition to diffusely in the cytoplasm. After bortezomib treatment the nuclear localization of p53β appeared more diffuse. No change in p53γ and p53β stability or subcellular localization was observed after exposure to chloroquine at the time points described above (data not shown).

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