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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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Tumor growth of H1299 p53β and H1299 p53γ cells.(A) H1299 in vivo s.c. tumors were measured in 3 independent experiments. In total: p53β+ tumors n = 17; p53γ+ tumors n = 17; Control vector (Ctrl; tdTomato+) n = 20. The tumor sizes of p53β+ and p53γ+ tumors were compared to ctrl tumors using Students t-test. * P-value<0.05, ** P-value<0.001, *** P-value<0.0001. Insert (top left corner) shows tumor measurements of tumors that were injected without matrigel. Error bars: standard error of mean. (B) Immunohistochemistry of p53 (DO-7) of s.c. tumors. Top: p53-negative vector control tumor (Ctrl). Middle: p53γ+ tumor. Bottom: p53β+ tumor. Scale bar left images 1 mm, right images 50 µm, middle insert 10 µm.
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pone-0056276-g006: Tumor growth of H1299 p53β and H1299 p53γ cells.(A) H1299 in vivo s.c. tumors were measured in 3 independent experiments. In total: p53β+ tumors n = 17; p53γ+ tumors n = 17; Control vector (Ctrl; tdTomato+) n = 20. The tumor sizes of p53β+ and p53γ+ tumors were compared to ctrl tumors using Students t-test. * P-value<0.05, ** P-value<0.001, *** P-value<0.0001. Insert (top left corner) shows tumor measurements of tumors that were injected without matrigel. Error bars: standard error of mean. (B) Immunohistochemistry of p53 (DO-7) of s.c. tumors. Top: p53-negative vector control tumor (Ctrl). Middle: p53γ+ tumor. Bottom: p53β+ tumor. Scale bar left images 1 mm, right images 50 µm, middle insert 10 µm.

Mentions: To examine the function of p53β and p53γ protein expression in a more realistic cancer environment exhibiting hypoxia and low nutrition, we examined the growth of subcutaneous H1299 in NSG mice. Surprisingly, an early tumor growth initiation followed by a significantly increased tumor growth was found of both p53γ and p53β congenic H1299 cells, compared to vector control (Figure 6A). This was in contrast to in vitro findings whereby the 3H-thymidine-incorporation assay showed only a minor decrease in proliferation of p53β+ and p53γ+ cells (Figure 2B). To evaluate if the growth factors present in the matrigel (for example TGF-β, epidermal growth factor, insulin-like growth factor and fibroblast growth factor) injected with the tumor cells could influence growth of p53β+ and p53γ+ tumors, the same assay was performed without matrigel. However, the same pattern was observed with these tumors (Figure 6A, top left graph). Immunohistochemistry of p53 in tumors (Figure 6B) reflected the previous in vitro observations by immunofluorescence (Figure 1C) demonstrating strong p53β signals (bottom images) with a major localization to the nucleus (insert), and p53γ signals of predominantly cytoplasmic origins.


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)

Tumor growth of H1299 p53β and H1299 p53γ cells.(A) H1299 in vivo s.c. tumors were measured in 3 independent experiments. In total: p53β+ tumors n = 17; p53γ+ tumors n = 17; Control vector (Ctrl; tdTomato+) n = 20. The tumor sizes of p53β+ and p53γ+ tumors were compared to ctrl tumors using Students t-test. * P-value<0.05, ** P-value<0.001, *** P-value<0.0001. Insert (top left corner) shows tumor measurements of tumors that were injected without matrigel. Error bars: standard error of mean. (B) Immunohistochemistry of p53 (DO-7) of s.c. tumors. Top: p53-negative vector control tumor (Ctrl). Middle: p53γ+ tumor. Bottom: p53β+ tumor. Scale bar left images 1 mm, right images 50 µm, middle insert 10 µm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3569410&req=5

pone-0056276-g006: Tumor growth of H1299 p53β and H1299 p53γ cells.(A) H1299 in vivo s.c. tumors were measured in 3 independent experiments. In total: p53β+ tumors n = 17; p53γ+ tumors n = 17; Control vector (Ctrl; tdTomato+) n = 20. The tumor sizes of p53β+ and p53γ+ tumors were compared to ctrl tumors using Students t-test. * P-value<0.05, ** P-value<0.001, *** P-value<0.0001. Insert (top left corner) shows tumor measurements of tumors that were injected without matrigel. Error bars: standard error of mean. (B) Immunohistochemistry of p53 (DO-7) of s.c. tumors. Top: p53-negative vector control tumor (Ctrl). Middle: p53γ+ tumor. Bottom: p53β+ tumor. Scale bar left images 1 mm, right images 50 µm, middle insert 10 µm.
Mentions: To examine the function of p53β and p53γ protein expression in a more realistic cancer environment exhibiting hypoxia and low nutrition, we examined the growth of subcutaneous H1299 in NSG mice. Surprisingly, an early tumor growth initiation followed by a significantly increased tumor growth was found of both p53γ and p53β congenic H1299 cells, compared to vector control (Figure 6A). This was in contrast to in vitro findings whereby the 3H-thymidine-incorporation assay showed only a minor decrease in proliferation of p53β+ and p53γ+ cells (Figure 2B). To evaluate if the growth factors present in the matrigel (for example TGF-β, epidermal growth factor, insulin-like growth factor and fibroblast growth factor) injected with the tumor cells could influence growth of p53β+ and p53γ+ tumors, the same assay was performed without matrigel. However, the same pattern was observed with these tumors (Figure 6A, top left graph). Immunohistochemistry of p53 in tumors (Figure 6B) reflected the previous in vitro observations by immunofluorescence (Figure 1C) demonstrating strong p53β signals (bottom images) with a major localization to the nucleus (insert), and p53γ signals of predominantly cytoplasmic origins.

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