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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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Basal characteristics of H1299 p53β and H1299 p53γ.(A) p53 immunoblot (Bp53-12) of H1299 cells transduced with p53β-tdTomato and p53γ-tdTomato construct. β-actin was used as loading control. (B) 3H-thymidine incorporation assay of H1299 p53β, H1299 p53γ and H1299 control cells (tdTomato vector control). The graph shows results from four individual experiments (average of 18 wells each). The experiments have been normalized. ** P-value<0.01. Error bars: Standard Error of the Mean (SEM). (C) Cell proliferation measured by Electric-Substrate Impedance Sensing. Impedance values are normalized after initial cell stabilization and shown as ratio of normalization value. The graph shows results from four measurements of vector control cells and six measurements of p53β+ and p53γ+ cells from two separate experiments. Standard error of mean is denoted by dotted lines. Highest variation in cell proliferation occurred after 30 hours after initiation, *P-value<0.05 calculated by paired Students t-test. (D) and (E) show immunoblot of basal level of Bax and p21, in both p53β+ and p53γ+ H1299 cells. GAPDH act as loading control, and the ratio of p21 or Bax to loading control with control value set to 1.0 is indicated. (F) Transient transfection of H1299 p53β and H1299 p53γ with the 13×p53RE-GFP construct and H1299 wt cells with both p53FL-construct and 13×p53RE-GFP were analyzed by flow cytometry (n = 2). Results are presented as a ratio of GFP positive cells in H1299 p53β, H1299 p53γ and H1299 cells transiently transfected with full-length p53 to H1299 vector control. Error bars: standard error of mean. Student's t-test give P-value 0.053 of p53β cells versus vector control, and P-value 0.21 of p53γ versus vector control.
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pone-0056276-g002: Basal characteristics of H1299 p53β and H1299 p53γ.(A) p53 immunoblot (Bp53-12) of H1299 cells transduced with p53β-tdTomato and p53γ-tdTomato construct. β-actin was used as loading control. (B) 3H-thymidine incorporation assay of H1299 p53β, H1299 p53γ and H1299 control cells (tdTomato vector control). The graph shows results from four individual experiments (average of 18 wells each). The experiments have been normalized. ** P-value<0.01. Error bars: Standard Error of the Mean (SEM). (C) Cell proliferation measured by Electric-Substrate Impedance Sensing. Impedance values are normalized after initial cell stabilization and shown as ratio of normalization value. The graph shows results from four measurements of vector control cells and six measurements of p53β+ and p53γ+ cells from two separate experiments. Standard error of mean is denoted by dotted lines. Highest variation in cell proliferation occurred after 30 hours after initiation, *P-value<0.05 calculated by paired Students t-test. (D) and (E) show immunoblot of basal level of Bax and p21, in both p53β+ and p53γ+ H1299 cells. GAPDH act as loading control, and the ratio of p21 or Bax to loading control with control value set to 1.0 is indicated. (F) Transient transfection of H1299 p53β and H1299 p53γ with the 13×p53RE-GFP construct and H1299 wt cells with both p53FL-construct and 13×p53RE-GFP were analyzed by flow cytometry (n = 2). Results are presented as a ratio of GFP positive cells in H1299 p53β, H1299 p53γ and H1299 cells transiently transfected with full-length p53 to H1299 vector control. Error bars: standard error of mean. Student's t-test give P-value 0.053 of p53β cells versus vector control, and P-value 0.21 of p53γ versus vector control.

Mentions: In order to study isoform specific biology, retroviral constructs/vectors containing p53β, p53γ or full-length p53 (Figure 1B) were generated to either retrovirally transduce or transiently transfect p53 cancer cell lines. The p53 lung carcinoma H1299 cell line was retrovirally transduced and FACS-sorted to generate stably expressing p53β, p53γ or full-length p53 H1299 cell lines. Employing a retroviral vector containing the p53 isoform and a fluorescent protein marker (tdTomato), transduction and sorting of tdTomato+ cells was performed twice (Figure 1B; see Materials and Methods for experimental details). Sorted cells were evaluated for tdTomato expression by flow cytometry and fluorescence microscopy (Figure 1C), and re-sorted for tdTomato expression if needed. Considerably fewer tdTomato+ fluorescent p53γ+ cells were observed following all transductions when compared to transduction efficiencies obtained with p53β+ cells (not shown). Full-length p53+ congenic H1299 cells could not be established, presumably due to the cytotoxic effect of p53 expression. p53 immunofluorescence showed a predominantly nuclear localization of p53β and both nuclear and cytoplasmic localization of p53γ (Figure 1C). p53β or p53γ in H1299 cells was confirmed by PCR of TP53 segment sequencing (exon 1–12) of both strands and immunoblot of p53 protein isoforms (Figure 2A; for details see Materials and Methods section). Immunoblot showed that p53β was expressed at considerably higher levels compared to p53γ (Figure 2A).


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)

Basal characteristics of H1299 p53β and H1299 p53γ.(A) p53 immunoblot (Bp53-12) of H1299 cells transduced with p53β-tdTomato and p53γ-tdTomato construct. β-actin was used as loading control. (B) 3H-thymidine incorporation assay of H1299 p53β, H1299 p53γ and H1299 control cells (tdTomato vector control). The graph shows results from four individual experiments (average of 18 wells each). The experiments have been normalized. ** P-value<0.01. Error bars: Standard Error of the Mean (SEM). (C) Cell proliferation measured by Electric-Substrate Impedance Sensing. Impedance values are normalized after initial cell stabilization and shown as ratio of normalization value. The graph shows results from four measurements of vector control cells and six measurements of p53β+ and p53γ+ cells from two separate experiments. Standard error of mean is denoted by dotted lines. Highest variation in cell proliferation occurred after 30 hours after initiation, *P-value<0.05 calculated by paired Students t-test. (D) and (E) show immunoblot of basal level of Bax and p21, in both p53β+ and p53γ+ H1299 cells. GAPDH act as loading control, and the ratio of p21 or Bax to loading control with control value set to 1.0 is indicated. (F) Transient transfection of H1299 p53β and H1299 p53γ with the 13×p53RE-GFP construct and H1299 wt cells with both p53FL-construct and 13×p53RE-GFP were analyzed by flow cytometry (n = 2). Results are presented as a ratio of GFP positive cells in H1299 p53β, H1299 p53γ and H1299 cells transiently transfected with full-length p53 to H1299 vector control. Error bars: standard error of mean. Student's t-test give P-value 0.053 of p53β cells versus vector control, and P-value 0.21 of p53γ versus vector control.
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Related In: Results  -  Collection

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pone-0056276-g002: Basal characteristics of H1299 p53β and H1299 p53γ.(A) p53 immunoblot (Bp53-12) of H1299 cells transduced with p53β-tdTomato and p53γ-tdTomato construct. β-actin was used as loading control. (B) 3H-thymidine incorporation assay of H1299 p53β, H1299 p53γ and H1299 control cells (tdTomato vector control). The graph shows results from four individual experiments (average of 18 wells each). The experiments have been normalized. ** P-value<0.01. Error bars: Standard Error of the Mean (SEM). (C) Cell proliferation measured by Electric-Substrate Impedance Sensing. Impedance values are normalized after initial cell stabilization and shown as ratio of normalization value. The graph shows results from four measurements of vector control cells and six measurements of p53β+ and p53γ+ cells from two separate experiments. Standard error of mean is denoted by dotted lines. Highest variation in cell proliferation occurred after 30 hours after initiation, *P-value<0.05 calculated by paired Students t-test. (D) and (E) show immunoblot of basal level of Bax and p21, in both p53β+ and p53γ+ H1299 cells. GAPDH act as loading control, and the ratio of p21 or Bax to loading control with control value set to 1.0 is indicated. (F) Transient transfection of H1299 p53β and H1299 p53γ with the 13×p53RE-GFP construct and H1299 wt cells with both p53FL-construct and 13×p53RE-GFP were analyzed by flow cytometry (n = 2). Results are presented as a ratio of GFP positive cells in H1299 p53β, H1299 p53γ and H1299 cells transiently transfected with full-length p53 to H1299 vector control. Error bars: standard error of mean. Student's t-test give P-value 0.053 of p53β cells versus vector control, and P-value 0.21 of p53γ versus vector control.
Mentions: In order to study isoform specific biology, retroviral constructs/vectors containing p53β, p53γ or full-length p53 (Figure 1B) were generated to either retrovirally transduce or transiently transfect p53 cancer cell lines. The p53 lung carcinoma H1299 cell line was retrovirally transduced and FACS-sorted to generate stably expressing p53β, p53γ or full-length p53 H1299 cell lines. Employing a retroviral vector containing the p53 isoform and a fluorescent protein marker (tdTomato), transduction and sorting of tdTomato+ cells was performed twice (Figure 1B; see Materials and Methods for experimental details). Sorted cells were evaluated for tdTomato expression by flow cytometry and fluorescence microscopy (Figure 1C), and re-sorted for tdTomato expression if needed. Considerably fewer tdTomato+ fluorescent p53γ+ cells were observed following all transductions when compared to transduction efficiencies obtained with p53β+ cells (not shown). Full-length p53+ congenic H1299 cells could not be established, presumably due to the cytotoxic effect of p53 expression. p53 immunofluorescence showed a predominantly nuclear localization of p53β and both nuclear and cytoplasmic localization of p53γ (Figure 1C). p53β or p53γ in H1299 cells was confirmed by PCR of TP53 segment sequencing (exon 1–12) of both strands and immunoblot of p53 protein isoforms (Figure 2A; for details see Materials and Methods section). Immunoblot showed that p53β was expressed at considerably higher levels compared to p53γ (Figure 2A).

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