Overexpressed TP73 induces apoptosis in medulloblastoma.
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.


CONCLUSIONS
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.
Castellino
Robert C
RC
Texas Children's Cancer Center, Department of Pediatrics, Baylor College of Medicine, Houston, TX 77030, USA. Craig_Castellino@oz.ped.emory.edu <Craig_Castellino@oz.ped.emory.edu>
De Bortoli
Massimiliano
M
Lin
Linda L
LL
Skapura
Darlene G
DG
Rajan
Jessen A
JA
Adesina
Adekunle M
AM
Perlaky
Laszlo
L
Irwin
Meredith S
MS
Kim
John Y H
JY
D017209:Apoptosis / Q000235:genetics*; D014408:Biomarkers, Tumor / Q000235:genetics; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000235:genetics; D002528:Cerebellar Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D002648:Child; D002675:Child, Preschool; D004268:DNA-Binding Proteins / Q000235:genetics*; D018450:Disease Progression; D005434:Flow Cytometry; D005500:Follow-Up Studies; D015972:Gene Expression Regulation, Neoplastic; D020868:Gene Silencing; D006801:Humans; D007150:Immunohistochemistry; D020287:In Situ Nick-End Labeling; D007223:Infant; D008297:Male; D008527:Medulloblastoma / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D009687:Nuclear Proteins / Q000235:genetics*; D011379:Prognosis; D012334:RNA, Neoplasm / Q000235:genetics*; D012189:Retrospective Studies; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012720:Severity of Illness Index; D013997:Time Factors; D025521:Tumor Suppressor Proteins / Q000235:genetics*
D014408:Biomarkers, Tumor; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D012334:RNA, Neoplasm; D025521:Tumor Suppressor Proteins