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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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p53β and p53γ protein modulation after treatment with camptothecin or doxorubicin.(A) Immunoblot analysis of p53 levels in H1299 p53γ and H1299 p53β cells after treatment with 0.5 µM doxorubicin (Dox) and 0.2 µM camptothecin (CPT) (8 hrs incubation time). Bar graphs represent the mean of four different experiments. * P-value<0.05, ** P-value<0.01. (B–G) Immunoblot analysis of Bax, p21, Chk1, Puma, Tigar and Mdm2 levels in H1299 p53β, H1299 p53γ and H1299 vector control cells after treatment with 0.5 µM Dox and 0.2 µM CPT (8 hrs). β-actin act as loading control, and the ratio of p53, Bax, p21,Chk1, Puma, Tigar or Mdm2 to loading control compared to value of untreated vector control cells (set to 1.0) is indicated. Bar graphs represent the mean of five (Bax), four (p21, Chk1, Tigar, Mdm2) or three (Puma) different experiments. Error bars: standard error of mean. Protein levels were compared using Student's t-test. * P-value<0.05, ** P-value<0.01. Brackets represent significant changes in basal protein levels relative to untreated H1299 vector control cells.
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pone-0056276-g004: p53β and p53γ protein modulation after treatment with camptothecin or doxorubicin.(A) Immunoblot analysis of p53 levels in H1299 p53γ and H1299 p53β cells after treatment with 0.5 µM doxorubicin (Dox) and 0.2 µM camptothecin (CPT) (8 hrs incubation time). Bar graphs represent the mean of four different experiments. * P-value<0.05, ** P-value<0.01. (B–G) Immunoblot analysis of Bax, p21, Chk1, Puma, Tigar and Mdm2 levels in H1299 p53β, H1299 p53γ and H1299 vector control cells after treatment with 0.5 µM Dox and 0.2 µM CPT (8 hrs). β-actin act as loading control, and the ratio of p53, Bax, p21,Chk1, Puma, Tigar or Mdm2 to loading control compared to value of untreated vector control cells (set to 1.0) is indicated. Bar graphs represent the mean of five (Bax), four (p21, Chk1, Tigar, Mdm2) or three (Puma) different experiments. Error bars: standard error of mean. Protein levels were compared using Student's t-test. * P-value<0.05, ** P-value<0.01. Brackets represent significant changes in basal protein levels relative to untreated H1299 vector control cells.

Mentions: Immunoblots of camptothecin- and doxorubicin-treated H1299 p53β, H1299 p53γ and H1299 vector control cells showed a significant decrease in p53γ and p53β expression indicating degradation of p53β and p53γ protein (Figure 4A). Camptothecin and doxorubicin treatment in H1299 p53β cells amplified the Bax and p21(CIP1/WAF1) response (Figure 4B,C). p53γ+ cells showed a significant decrease in Bax after doxorubicin treatment while p21(CIP1/WAF1) was significantly elevated after camptothecin treatment (Figure 4B,C). Furthermore, an increase in basal level of the kinase Chk1 was detected with p53β. Chk1 was significantly reduced upon camptothecin treatment (Figure 4D) and no clear changes in Puma protein levels were seen between the groups (Figure 4E). Decreased protein levels of Tigar (TP53-induced glycolysis and apoptosis factor) protein in p53β+ and p53γ+ cells after doxorubicin treatment were observed (Figure 4F). Increased basal Mdm2 levels were observed in both p53β and p53γ, and reduced in all cells after chemotherapy treatments (Figure 4G).


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)

p53β and p53γ protein modulation after treatment with camptothecin or doxorubicin.(A) Immunoblot analysis of p53 levels in H1299 p53γ and H1299 p53β cells after treatment with 0.5 µM doxorubicin (Dox) and 0.2 µM camptothecin (CPT) (8 hrs incubation time). Bar graphs represent the mean of four different experiments. * P-value<0.05, ** P-value<0.01. (B–G) Immunoblot analysis of Bax, p21, Chk1, Puma, Tigar and Mdm2 levels in H1299 p53β, H1299 p53γ and H1299 vector control cells after treatment with 0.5 µM Dox and 0.2 µM CPT (8 hrs). β-actin act as loading control, and the ratio of p53, Bax, p21,Chk1, Puma, Tigar or Mdm2 to loading control compared to value of untreated vector control cells (set to 1.0) is indicated. Bar graphs represent the mean of five (Bax), four (p21, Chk1, Tigar, Mdm2) or three (Puma) different experiments. Error bars: standard error of mean. Protein levels were compared using Student's t-test. * P-value<0.05, ** P-value<0.01. Brackets represent significant changes in basal protein levels relative to untreated H1299 vector control cells.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3569410&req=5

pone-0056276-g004: p53β and p53γ protein modulation after treatment with camptothecin or doxorubicin.(A) Immunoblot analysis of p53 levels in H1299 p53γ and H1299 p53β cells after treatment with 0.5 µM doxorubicin (Dox) and 0.2 µM camptothecin (CPT) (8 hrs incubation time). Bar graphs represent the mean of four different experiments. * P-value<0.05, ** P-value<0.01. (B–G) Immunoblot analysis of Bax, p21, Chk1, Puma, Tigar and Mdm2 levels in H1299 p53β, H1299 p53γ and H1299 vector control cells after treatment with 0.5 µM Dox and 0.2 µM CPT (8 hrs). β-actin act as loading control, and the ratio of p53, Bax, p21,Chk1, Puma, Tigar or Mdm2 to loading control compared to value of untreated vector control cells (set to 1.0) is indicated. Bar graphs represent the mean of five (Bax), four (p21, Chk1, Tigar, Mdm2) or three (Puma) different experiments. Error bars: standard error of mean. Protein levels were compared using Student's t-test. * P-value<0.05, ** P-value<0.01. Brackets represent significant changes in basal protein levels relative to untreated H1299 vector control cells.
Mentions: Immunoblots of camptothecin- and doxorubicin-treated H1299 p53β, H1299 p53γ and H1299 vector control cells showed a significant decrease in p53γ and p53β expression indicating degradation of p53β and p53γ protein (Figure 4A). Camptothecin and doxorubicin treatment in H1299 p53β cells amplified the Bax and p21(CIP1/WAF1) response (Figure 4B,C). p53γ+ cells showed a significant decrease in Bax after doxorubicin treatment while p21(CIP1/WAF1) was significantly elevated after camptothecin treatment (Figure 4B,C). Furthermore, an increase in basal level of the kinase Chk1 was detected with p53β. Chk1 was significantly reduced upon camptothecin treatment (Figure 4D) and no clear changes in Puma protein levels were seen between the groups (Figure 4E). Decreased protein levels of Tigar (TP53-induced glycolysis and apoptosis factor) protein in p53β+ and p53γ+ cells after doxorubicin treatment were observed (Figure 4F). Increased basal Mdm2 levels were observed in both p53β and p53γ, and reduced in all cells after chemotherapy treatments (Figure 4G).

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