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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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H1299 colony formation and proliferation after chemotherapy.(A) Colony formation assay of H1299 p53β, H1299 p53γ and H1299 vector control demonstrated significantly decreased colony formation in p53β+ and p53γ+ cells after seven-days treatment with 25 nM doxorubicin (Dox). Insert shows a magnification of selected colonies from the doxorubicin treated plates. (B) illustrates count of treated colonies relative to colony count of untreated cells from (A). Controls within each group have been normalized to 100 colonies, and the treatment groups have been subsequently adjusted. (C) Each bar represents the number of colonies within each treatment group, and statistics have been calculated based on the untreated control within each cell subtype (p53β, p53γ, wt (not transduced) or vector control (ctrl, tdTomato vector control). (D) 3H-thymidine incorporation assay of p53β+ and p53γ+ cells exposed for 8 hrs to 0.5 µM Dox (n = 6 experiments), 0.4 µM camptothecin (CPT; n = 2), or vehicle (DMSO) (each separate experiments have six parallels each). Cytarabine (AraC) at 0.1 µM gave no significant response. Columns represent the ratio of treated to control (DMSO) 3H-thymidine uptake. * P-value<0.05, **P-value<0.01, *** P-value<0.001. Error bars: standard error of mean.
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pone-0056276-g003: H1299 colony formation and proliferation after chemotherapy.(A) Colony formation assay of H1299 p53β, H1299 p53γ and H1299 vector control demonstrated significantly decreased colony formation in p53β+ and p53γ+ cells after seven-days treatment with 25 nM doxorubicin (Dox). Insert shows a magnification of selected colonies from the doxorubicin treated plates. (B) illustrates count of treated colonies relative to colony count of untreated cells from (A). Controls within each group have been normalized to 100 colonies, and the treatment groups have been subsequently adjusted. (C) Each bar represents the number of colonies within each treatment group, and statistics have been calculated based on the untreated control within each cell subtype (p53β, p53γ, wt (not transduced) or vector control (ctrl, tdTomato vector control). (D) 3H-thymidine incorporation assay of p53β+ and p53γ+ cells exposed for 8 hrs to 0.5 µM Dox (n = 6 experiments), 0.4 µM camptothecin (CPT; n = 2), or vehicle (DMSO) (each separate experiments have six parallels each). Cytarabine (AraC) at 0.1 µM gave no significant response. Columns represent the ratio of treated to control (DMSO) 3H-thymidine uptake. * P-value<0.05, **P-value<0.01, *** P-value<0.001. Error bars: standard error of mean.

Mentions: Following the observation that the isoforms may activate p53-responsive genes, we examined the response of the H1299 p53β and H1299 p53γ cell lines to chemotherapeutics with a colony-formation assay. The pyrimidine antagonist arabinofuranosyl cytidine (cytarabine, Ara-C), the cytotoxic antibiotic and topoisomerase II inhibitor doxorubicin (Dox) and the topoisomerase I inhibitor camptothecin (CPT) were tested. In both H1299 p53β and H1299 p53γ, a significantly decreased colony formation compared to vector control was observed when treated with doxorubicin (Figure 3A–C). Treatment with Ara-C and camptothecin (not shown) showed less effect on colony formation than doxorubicin, but nevertheless revealed a tendency towards reduced number of colonies. Decreased proliferation of H1299 p53β and H1299 p53γ after treatment with doxorubicin and camptothecin (especially at higher dose) was also identified by a 3H-thymidine incorporation assay (Figure 3D). Transient transfection of p53 SAOS-2 osteosarcoma cell line with p53β- and p53γ-tdTomato construct followed by treatment with 0.5 µM doxorubicin for 24 hours, also showed significant reduced 3H-thymidine DNA incorporation in p53β+ cells (data not shown). This was not seen with the p53γ or full-length p53, and we propose that this lack of doxorubicin toxicity may be caused by high cell death caused by the cell death induction potential of p53γ or full-length p53.


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)

H1299 colony formation and proliferation after chemotherapy.(A) Colony formation assay of H1299 p53β, H1299 p53γ and H1299 vector control demonstrated significantly decreased colony formation in p53β+ and p53γ+ cells after seven-days treatment with 25 nM doxorubicin (Dox). Insert shows a magnification of selected colonies from the doxorubicin treated plates. (B) illustrates count of treated colonies relative to colony count of untreated cells from (A). Controls within each group have been normalized to 100 colonies, and the treatment groups have been subsequently adjusted. (C) Each bar represents the number of colonies within each treatment group, and statistics have been calculated based on the untreated control within each cell subtype (p53β, p53γ, wt (not transduced) or vector control (ctrl, tdTomato vector control). (D) 3H-thymidine incorporation assay of p53β+ and p53γ+ cells exposed for 8 hrs to 0.5 µM Dox (n = 6 experiments), 0.4 µM camptothecin (CPT; n = 2), or vehicle (DMSO) (each separate experiments have six parallels each). Cytarabine (AraC) at 0.1 µM gave no significant response. Columns represent the ratio of treated to control (DMSO) 3H-thymidine uptake. * P-value<0.05, **P-value<0.01, *** P-value<0.001. Error bars: standard error of mean.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3569410&req=5

pone-0056276-g003: H1299 colony formation and proliferation after chemotherapy.(A) Colony formation assay of H1299 p53β, H1299 p53γ and H1299 vector control demonstrated significantly decreased colony formation in p53β+ and p53γ+ cells after seven-days treatment with 25 nM doxorubicin (Dox). Insert shows a magnification of selected colonies from the doxorubicin treated plates. (B) illustrates count of treated colonies relative to colony count of untreated cells from (A). Controls within each group have been normalized to 100 colonies, and the treatment groups have been subsequently adjusted. (C) Each bar represents the number of colonies within each treatment group, and statistics have been calculated based on the untreated control within each cell subtype (p53β, p53γ, wt (not transduced) or vector control (ctrl, tdTomato vector control). (D) 3H-thymidine incorporation assay of p53β+ and p53γ+ cells exposed for 8 hrs to 0.5 µM Dox (n = 6 experiments), 0.4 µM camptothecin (CPT; n = 2), or vehicle (DMSO) (each separate experiments have six parallels each). Cytarabine (AraC) at 0.1 µM gave no significant response. Columns represent the ratio of treated to control (DMSO) 3H-thymidine uptake. * P-value<0.05, **P-value<0.01, *** P-value<0.001. Error bars: standard error of mean.
Mentions: Following the observation that the isoforms may activate p53-responsive genes, we examined the response of the H1299 p53β and H1299 p53γ cell lines to chemotherapeutics with a colony-formation assay. The pyrimidine antagonist arabinofuranosyl cytidine (cytarabine, Ara-C), the cytotoxic antibiotic and topoisomerase II inhibitor doxorubicin (Dox) and the topoisomerase I inhibitor camptothecin (CPT) were tested. In both H1299 p53β and H1299 p53γ, a significantly decreased colony formation compared to vector control was observed when treated with doxorubicin (Figure 3A–C). Treatment with Ara-C and camptothecin (not shown) showed less effect on colony formation than doxorubicin, but nevertheless revealed a tendency towards reduced number of colonies. Decreased proliferation of H1299 p53β and H1299 p53γ after treatment with doxorubicin and camptothecin (especially at higher dose) was also identified by a 3H-thymidine incorporation assay (Figure 3D). Transient transfection of p53 SAOS-2 osteosarcoma cell line with p53β- and p53γ-tdTomato construct followed by treatment with 0.5 µM doxorubicin for 24 hours, also showed significant reduced 3H-thymidine DNA incorporation in p53β+ cells (data not shown). This was not seen with the p53γ or full-length p53, and we propose that this lack of doxorubicin toxicity may be caused by high cell death caused by the cell death induction potential of p53γ or full-length p53.

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