[CANCER RESEARCH 54, 1098-111~4. Fcbru:lrv 15. It~941 Defective Human Retinoblastoma Protein Identified by Lack of Interaction with the E1A Oncoprotein I Marco G. Paggi, z Fabio MarteUi, Maurizio Fanciulli, Armando Felsani, Salvatore Sciacchitano, Marco Varmi, Tiziana Bruno, Carmine M. Carapella, and Aristide Floridi Laboratori di Metabolismo Celhdare e Farmacocinetica [M. G. P., M. E, M. V, T. B., A. FI.]; Biologia Moh'cohu'e [F. M., A. Fe.], Centtv Ricerca Sperimentah', Istituto Regina Elena per lo Studio e la Cura dei Tumori, Via delle Messi d'Oro, 156, O0158 Rome; Istituto Tecnologie Biomediche, C. N. R., Via Morgagni 30/E, 00161 Rome [A. Fe.]; Dipartimento Medicina Sperimentale, Universith "La Sapienza, '" Viale Regina Elena, 361, 00161 Rome [S. S.]: and Divisione di Neurochirurgia, lstituto Regina Elena per lo Studio e la Cura dei Tumori, Viale Regina Elena, 291, 00161 Rome,/C. M. C.], Italy ABSTRACT The retinoblastoma gene product, pRb, is a nuclear phosphoprotein with DNA-binding property (21, 22). Normal pRb shows a certain Inactivating mutations of the retinoblastoma susceptibility gene (Rb) microheterogeneity in SDS-PAGE, due to different degrees of phos- are involved in the pathogenesis of hereditary and sporadic retinoblas- phorylation, so that the protein is usually found between 105 and 114 toma. Alterations in the Rb gene have also been found in several other human tumors occurring with epidemiological incidence higher than that kDa of apparent molecular mass. The phosphorylation status of pRb of retinoblastoma. Four human malignant glioma cell lines were examined oscillates between an unphosphorylated or an underphosphorylated for abnormalities in the retinoblastoma gene product (pRb), using a pro- form (fast-migrating), during the Go-G~ phases of the cell cycle, and cedure based on the interaction of pRb with an in vitro-translated adeno- a fully phosphorylated form (slow-migrating), when the cell reaches virus E1A oncoprotein. In the CRS-A2 cell line, derived from a glioblas- the Gt-S boundary (4-6, 23). This microheterogeneous pattern is an toma muitiforme, pRb did not bind with the in vitro-translated E1A index of asynchronism in the cell population and is the most common protein. Restriction analysis of the CRS-A2 Rb gene and Rb mRNA ex- behavior, either in vivo or in vitro (4, 24). pression provided patterns that could not be distinguished from the other The pRb is not detected in some tumor cell lines, even when glioma cell lines. Further investigation revealed the presence of a trun- Southern and Northern blot analyses provide a pattern that cannot be cated pRb in the CRS-A2 cell line, due to a nucleotide insertion in the distinguished from the normal one (17, 22). Reinsertion of wild type coding sequence at position 2550. In addition, this truncated Rb protein was indetectable in phosphorylated form. The binding assay with the Rb gene in human cancer cell lines with proven pRb impairment in vitro-translated E1A was also used to study other cell lines with reverts the neoplastic phenotype (16, 25, 26), thus ultimately confirm- known mutations in the Rb gene. This method, which evaluates the inter- ing the oncosuppressor role of Rb. action between in vitro.translated E1A and the pRb, is proposed as a The Rb gene product is capable of interacting with oncoproteins rapid screening for detecting functional alterations in the retinoblastoma from DNA tumor viruses, such as the adenovirus EIA (24), the SV40 protein. large T (27, 28), and the human papillomavirus E7 (29). This finding supports the hypothesis that these viruses can induce transformation INTRODUCTION by neutralizing the cellular mechanisms involved in growth suppres- sion (24). The E1A-binding site of the pRb molecule has been also The retinoblastoma susceptibility gene (Rb) is the prototype for a implicated in the interaction with the other DNA virus oncoproteins class of genes the inactivation of which appears to be causally related (30) as well as with endogenous factors, such as E2E involved in to cancer; these genes are therefore referred to as tumor suppressor important functions in cellular proliferation control mechanisms (30- genes (1-3). Although the precise function of pRb is still unknown, its 32). Phosphorylated pRb interacts less strictly with viral oncoproteins activity is related to DNA synthesis (4, 5) or cell cycle regulation (6). (24, 28, 33) and releases bound E2F which, in its free form, can Gross alterations in the Rb gene have been shown to occur regularly activate transcription (34). either in sporadic or in inherited forms of retinoblastoma, where A simple method was designed for the functional screening of the deletion of chromosome 13 segments, containing the Rb locus, are Rb gene product, based on its interaction with the E1A oncoprotein. commonly reported (7, 8). Alterations in the Rb gene structure, lead- Briefly, IVT-E1A was challenged with tumor cell lysates, to evaluate ing to either loss or functional inactivation of the gene product, have its binding with pRb by means of a coimmunoprecipitation assay, been observed in more common human tumors such as small cell lung using an anti-pRb monoclonal antibody. The method described in this cancer (9, 10), breast cancer (11, 12), osteogenic sarcomas (13, 14), paper permitted identification of a defective Rb protein in the CRS-A2 leukemias (15), prostate (16) and bladder (17, 18) carcinomas, and cell line, isolated in our laboratory starting from a surgical specimen malignant gliomas (19, 20). Southern blot analysis of tumor DNA of a glioblastoma multiforme. Further investigation revealed the pres- probed with Rb cDNA 3 is often not a sensitive enough method to ence of a nucleotide insertion in the CRS-A2 malignant glioma at provide evidence of such subtle alterations (17). Even the Rb-specific position 2550 of the Rb coding sequence. mRNA evaluation in Northern blot analysis does not seem to consis- tently assess the structural and functional status of Rb (17). MATERIALS AND METHODS Cell Culture Received 6/14/93; accepted 12/16/93. The costs of publication of this article were defrayed in part by the payment of page The LI (35-37), DF (37) (courtesy of Dr. G. Zupi, Istituto Regina Elena, charges. This article must therefore be hereby marked advertisement in accordance with Rome, Italy), and ADF (courtesy of Dr. W. Malorni, Istituto Superiore di 18 U.S.C. Section 1734 solely to indicate this fact. Sanit'~, Rome, Italy) human malignant glioma cells; Y79 (38) and WERI-Rbl 1 This work was supported by the Progetto Finalizzato "Applicazioni Cliniche della Ricerca Oncologica" and "Ingegneria Genetica," CNR, Rome, Italy, and by the "Asso- (39) retinoblastomas (both courtesy of Dr. A. Albini, Istituto Nazionale Tumori, ciazione ltaliana per la Ricerca sul Cancro" (AIRC), Milan, Italy. F. M. is an AIRC Genoa, Italy); and H209 SCLC cells (40) were grown in RPMI 1640 (Gibco) postdoctoral fellow. CRS-A2 cells, established in 1991 in our Institute from a surgical specimen of 2 To whom requests for reprints should be addressed. a glioblastoma multiforme, were grown in Dulbecco's modified Eagle's 3 The abbreviations used are: cDNA, complementary DNA; DOC, sodium deoxycho- late; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; 1VT-E1A, medium/Ham's F-12 medium (Gibco). SAOS-2 osteosarcoma cells (41) (cour- in vitro-translated adenovirus EIA protein; PCR, polymerase chain reaction; DGGE, tesy of Dr. M. Zamanian, National Institute for Medical Research, London, denaturing gradient gel electrophoresis; nt, nucleotide. United Kingdom) and 293 cells (42) were grown in Dulbecco's modified 1098 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1994 American Association for Cancer Research. ABERRANT pRb IN A HUMAN MALIGNANT GIAOMA Eagle's medium (Gibco). Media were supplemented with 10% fetal calf serum priming technique (48). After a final high-stringency wash [0.1 • standard (15% for Y79 and WERI-Rbl cells), 2 mM glutamine, and antibiotics. Cells saline-citrate (1 • is 150 mM NaCI-15 mM citric acid, pH 7.0)-0.1% SDS at were incubated at 37~ in a 5% CO2 atmosphere. 60~ for 30 rain], the nitrocellulose was exposed at -70~ using Kodak XAR-5 film with Du Pont Cronex intensifying screens. Cell Labeling and Lysis Northern Blot Analysis Labeling with [3SS]Methionine. Subconfluent cell cultures were washed twice with prewarmed 150 mM NaCI-10 mM sodium phosphate, pH 7.20, and Total RNA from subconfluent cell cultures of each cell line was prepared incubated at 37~ in prewarmed methionine-free medium, plus 5% dialyzed according to the method of Chomczynsky and Sacchi (49) and resolved by fetal calf serum (Gibco) and 2 mM glutamine (6 ml of medium for each 75-cm 2 electrophoresis through 1.2% agarose gel in the presence of formaldehyde (47). flask) for 180 rain. The medium was then substituted with the same prewarmed Ten/xg of total RNA were loaded onto each lane. After transfer to nitrocellu- medium (2.5 ml for each 75-cm 2 flask) plus 9.25 MBq (250 /xCi) of [3sS]- lose filters (BA-85), hybridization was carried out with a human Rb cDNA methionine (Tran35S-Label; ICN) and the incubation was carried out for 180 probe (24) labeled with a random priming technique (48). After a final high rain at 37~ Cells were then washed twice in ice-cold 150 mM NaCI-10 mM stringency wash (0.2 x standard saline-citrate-0.1% SDS at 65~ for 30 min), sodium phosphate, pH 7.20, and lysed in ice-cold lysis buffer (150 mM the nitrocellulose was exposed at -70~ using Kodak XAR-5 film with Du NaC1-50 mM Tris-HCl, pH 8.00-5 mM EDTA-I% Nonidet P-40-1 mM PMSF-1 Pont Cronex intensifying screens.