pRb2/p130 promotes radiation-induced cell death in the glioblastoma cell line HJC12 by p73 upregulation and Bcl-2 downregulation Bruna Pucci 1 , Pier Paolo Claudio 2,3 , Valeria Masciullo 1 , Lorenza Bellincampi 4 , Alessandro Terrinoni 5 , Kamel Khalili 6 , Gerry Melino 5 and Antonio Giordano* ,1,2 1 Department of Pathology, Anatomy and Cell Biology, Jefferson Medical College, Philadelphia, Pennsylvania, PA 19107, USA; 2 Sbarro Institute for Cancer Research and Molecular Medicine, College of Science and Technology, Temple University, Philadelphia, Pennsylvania, PA 19122, USA; 3 Dipartimento di Scienze Odontostomatologiche e Maxillo-facciali, Universita’ di Napoli ‘Federico II’, Naples, 80131, Italy; 4 Dipartimento di Medicina Interna Universita’ di Roma ‘Tor Vergata’, Rome, 00133, Italy; 5 Laboratorio di Biochimica, Istituto Dermopatico dell’Immacolata (IDI-IRCCS) c/o Dipartimento di Medicina Sperimentale e Scienze Biochimiche, Universita’ di Roma ‘Tor Vergata’, Rome, 00133, Italy; 6 Center for Neuro Virology and Cancer Biology, Temple University, Philadelphia, Pennsylvania, PA 19122, USA This study shows that in the glioblastoma hamster cell line HJC12 the retinoblastoma family member pRb2/ p130 enhances g-radiation-induced cell death. In HJC12 cells the tetracycline-regulated expression of pRb2/p130 increased the percentage of g-radiation-induced apoptotic cells from 27 to 47%. pRb2/p130 overexpression was associated with the downregulation of the anti-apoptotic factor Bcl-2 and the upregulation of the steady-state protein levels of the pro-apoptotic transcription factor p73. In particular, RT – PCR showed a significant increase in the expression of the p73d isoform when pRb2/p130 was overexpressed. The ability of pRb2/p130 to modulate apoptosis was not associated with its role in mediating G0/G1 arrest during cell cycle progression. Our data suggest a role for pRb2/p130 in glioblastoma g-radiation-induced cell death, indicating that the antitumoral action of pRb2/p130 can regulate both inhibition of cell cycle progression and induction of cell death. Oncogene (2002) 21, 5897 – 5905. doi:10.1038/sj.onc. 1205750 Keywords: apoptosis; g-radiation; pRb2/p130; glioblas- toma; p73; Bcl-2; cell cycle Introduction pRb2/p130, originally identified in our laboratory, is a member of the retinoblastoma family along with pRb/ p105 and p107 (Stiegler and Giordano, 1998). Similarly to its homologues, pRb2/p130 is a tumor suppressor that negatively regulates cell cycle progression. As a member of the retinoblastoma family, pRb2/p130 interacts with cyclin-dependent kinases (cdks) and binds E2F transcription factors, in particular E2F-4, and inhibits S phase gene transcription. pRb2/p130 is regulated by cdks through phosphorylation. Hyper- phosphorylated pRb2/p130 loses its ability to inhibit E2F-4 transcription factors and to block cell cycle progression. Upon dephosphorylation, pRb2/p130 is activated and induces growth arrest at the G0/G1 phase of the cell cycle. pRb2/p130 is mainly expressed and active in arrested or differentiated cells (Mac- Lachlan et al., 1995). Increasing evidence has indicated that Rb family proteins, including pRb/p105, are not only involved in proliferation and transformation but also in apoptosis. Indeed, pRb/p105 has a protective function against apoptosis. In vivo studies show that pRb/p105 knock- out mice die in utero at 14 – 15 days with defects in the haematopoietic system and impaired development of the central and peripheral nervous system because of massive cell death (Jacks et al., 1992). Inappropri- ate cell death is also present in the liver, lens and skeletal muscle precursors. In vitro experiments confirm the results obtained with transgenic mice. TGF-b1 causes cell death by suppressing the expres- sion of pRb/p105 and by phosphorylating it (Fan et al., 1996). Similarly, IFNg-induced apoptosis is blocked in pRb/p105-positive cells (Berry et al., 1996). Restoring the function of pRb/p105 in a pRb/105-null osteosarcoma cell line inhibits irradia- tion-induced apoptosis (Haas-Kogan et al., 1995). Additional support for a protective role for pRb/ p105 against apoptosis comes from the observation that pRb/p105 is cleaved by caspases during apoptosis (An and Dou, 1996; Dou et al., 1997; Janicke et al., 1996). Not many data are currently available on the role of the other two members of the Rb family, pRb2/p130 and p107, in apoptosis. In the present study, we examine the role of pRb2/ p130 in g-radiation-induced apoptosis by using the hamster glioblastoma cell line HJC12, which was irradiated in the presence of tetracycline-regulated pRb2/p130 expression. Received 19 April 2002; revised 24 May 2002; accepted 7 June 2002 *Correspondence: A Giordano, Sbarro Institute for Cancer Research and Molecular Medicine, College of Science and Technology, Temple University, BioLife Science Building, Suite 333 1900 N 12th Street, Philadelphia, PA 19122, USA; E-mail: giordano@temple.edu Oncogene (2002) 21, 5897 – 5905 ª 2002 Nature Publishing Group All rights reserved 0950 – 9232/02 $25.00 www.nature.com/onc