Overexpressed TP73 induces apoptosis in medulloblastoma

Abstract

Background: Medulloblastoma is the most common malignant brain tumor of childhood. Children who relapse usually die of their disease, which reflects resistance to radiation and/or chemotherapy. Improvements in outcome require a better understanding of the molecular basis of medulloblastoma growth and treatment response. TP73 is a member of the TP53 tumor suppressor gene family that has been found to be overexpressed in a variety of tumors and mediates apoptotic responses to genotoxic stress. In this study, we assessed expression of TP73 RNA species in patient tumor specimens and in medulloblastoma cell lines, and manipulated expression of full-length TAp73 and amino-terminal truncated DeltaNp73 to assess their effects on growth.

Methods: We analyzed medulloblastoma samples from thirty-four pediatric patients and the established medulloblastoma cell lines, Daoy and D283MED, for expression of TP73 RNA including the full-length transcript and the 5'-terminal variants that encode the DeltaNp73 isoform, as well as TP53 RNA using quantitative real time-RTPCR. Protein expression of TAp73 and DeltaNp73 was quantitated with immunoblotting methods. Clinical outcome was analyzed based on TP73 RNA and p53 protein expression. To determine effects of overexpression or knock-down of TAp73 and DeltaNp73 on cell cycle and apoptosis, we analyzed transiently transfected medulloblastoma cell lines with flow cytometric and TUNEL methods.

Results: Patient medulloblastoma samples and cell lines expressed full-length and 5'-terminal variant TP73 RNA species in 100-fold excess compared to non-neoplastic brain controls. Western immunoblot analysis confirmed their elevated levels of TAp73 and amino-terminal truncated DeltaNp73 proteins. Kaplan-Meier analysis revealed trends toward favorable overall and progression-free survival of patients whose tumors display TAp73 RNA overexpression. Overexpression of TAp73 or DeltaNp73 induced apoptosis under basal growth conditions in vitro and sensitized them to cell death in response to chemotherapeutic agents.

Conclusion: These results indicate that primary medulloblastomas express significant levels of TP73 isoforms, and suggest that they can modulate the survival and genotoxic responsiveness of medulloblastomas cells.

Figure 1

Primary medulloblastoma specimens and medulloblastoma cell lines overexpress TAp73 and ΔNp73 RNA species relative to TP53. (A) TP73 gene and relative location of isoform-specific primers for qRT-RTPCR of TP73. Exons are depicted as boxes with overlying arrows corresponding to location of primers for PCR. (B) Established medulloblastoma cell lines, Daoy and D283MED (D283), and human adult cerebellum (Cbl) express high levels of TAp73 and amino-terminal splice variants encoding ΔNp73 in comparison to human fetal brain. Primary medulloblastoma (MB) samples from patients (n = 34) display similar overexpression of TAp73 and ΔNp73 RNA species. (C) Primary medulloblastoma samples from patients (n = 34) display overexpression of TAp73 and amino-terminal truncated TP73 RNA variants, relative to human fetal brain and normalized to GAPDH expression. By comparison, TP53 RNA is relatively underexpressed. Total ΔNp73 represents the sum of expression of all amino-terminal-truncated TP73 RNA variants (ΔNp73, ΔN'p73, ΔEx2p73, and ΔEx2/3p73). Columns, mean expression of at least 2 experiments; error bars, ± S.E.M. Y-axis, RNA expression relative to human fetal brain and normalized to GAPDH expression (N.B. log-scale).

Figure 2

Primary medulloblastoma specimens and medulloblastoma cell lines overexpress TAp73 and ΔNp73 proteins. Western immunoblot analysis of p53, TAp73, and ΔNp73 protein expression in: (A) established medulloblastoma cell lines, Daoy and D283MED (D283), and (B) primary medulloblastoma specimens. Immunoblots for p53 (FL-393) or p73 proteins (H-79, GC-15, and IMG-313) were stripped and re-probed for β-actin (C-2) to control for protein loading. Columns marked with numbers 1–8 represent Western blots of eight different primary medulloblastoma specimens. Additional bands presumably represent additional carboxy-terminal isoforms of TAp73 and ΔNp73. Relative migration of molecular weight standards is shown at left. Shown are representative blots from at least 3 experiments, normalized to ß-actin (mean ± S.E.M.). (C) Primary medulloblastoma (MB) specimens and medulloblastoma cell lines display significant expression of TAp73, ΔNp73, and p53 proteins as quantitated on Western blots. Shown are representative blots from at least 3 experiments, normalized to ß-actin (mean ± S.E.M.).

Figure 3

Intensity of p53 immunostaining correlates with overall survival. Examples of medulloblastoma sections displaying (A) 1+, (B) 2+, and (C) 3+ intensity of p53 immunostaining. (D) Kaplan-Meier survival analysis illustrating association of 2+ and 3+ intensity with adverse progression-free survival (p < 0.035 by logrank testing).

Figure 4

Medulloblastoma survival is associated with TAp73 overexpression. Kaplan-Meier analysis reveals trends toward adverse outcome associated with (A) age less than 36 months (p < 0.126 by logrank), and (B) metastatic (M+) disease at diagnosis (p < 0.024); while revealing (C) a trend toward better PFS (p < 0.052) and (D) better OS and in patients whose tumors display higher TAp73 RNA levels (p < 0.070). Patients were stratified (High vs. Low) according to tumor TAp73 levels, normalized to GAPDH and relative to fetal brain control. High = greater than or equal to the median tumor expression of TAp73 RNA. Low = less than the median tumor expression of TAp73 RNA.

Figure 5

Genotoxic agents induce TP73 expression and apoptosis in medulloblastoma cell lines. (A) Chemotherapeutic agents (cisplatin (CDDP) 1 µM, etoposide (VP-16) 10 µM, and doxorubicin (DOXO) 1 µM) induce TP73 expression in Daoy cells, especially ΔN’p73 species, as determined by qRT-RTPCR normalized to GAPDH expression. (B) CDDP-treated (1, 5, and 25 µM) D283 cells display induction of TP53 and TP73 RNA expression (including ΔN’p73 species) - similar to results observed in CDDP-treated Daoy cells. Western immunoblots reveal that (C) Daoy cells and (D) D283 cells transiently transfected with p53-GFP expression plasmid increase expression of tagged p53-GFP protein (at a higher apparent molecular weight than native p53), as well as the p53/p73 target, p21^Waf1(arrows, upper panels). Treatment with etoposide (VP-16, 1.5 µM) stabilizes wild-type p53 protein. Transient transfection with either TAp73 or ΔNp73 expression plasmids also increased respective protein levels, normalized to ß-actin loading control. (arrows lower panels).

Figure 6

TAp73 induces apoptosis of medulloblastoma cell lines. Histograms summarizing flow cytometric analysis of cell cycle distributions of: (A) transfected Daoy cell line, illustrating the sub-G0/G1 peak representing apoptotic nuclei; and (B) transfected D283 cell line in sub-G0/G1, with plasmids and treatment with VP-16 (1.5 µM), as indicated. Y-axis, % of cells with apoptotic features by flow cytometry; X-axis, transfected plasmid(s) and culture conditions. Western immunoblots also reveal that (C) Daoy cells and (D) D283 cells transiently transfected with isoform-specific siRNA reveal knockdown of their respective protein levels. D283 knockdowns reduced protein expression of the p53/p73 target, p21^Waf1, and cleavage of PARP (arrows).