Defective Human Retinoblastoma Protein Identified by Lack of Interaction with the E1A Oncoprotein I

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[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 suppression (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

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. Normalization was obtained using a glyc- p~g/ml aprotinin-1 /xg/ml leupeptin). One ml of the lysis buffer was added to eraidehyde-3-phosphate dehydrogenase cDNA probe. each 75-cm 2 flask; when indicated, 0.1% SDS and 0.5% DOC were added to the lysis buffer. Flasks were placed on ice for 30 min with occasional shaking. In Vitro Transcription and Translation of Adenovirus 5 E1A Cell lysates were then collected in 1.5-ml centrifuge tubes and spun for 10 min at 14,000 rpm in an Eppendorf 5415 centrifuge at 4~ Supernatants were The adenovirus EIA mRNA was synthesized in vitro using a plasmid collected for immunoprecipitation. containing the HindIII-BamHI fragment of pMTEB12S (50) cloned in the Labeling with [32p]Orthophosphoric Acid. The procedure was similar to pSP64 vector cut with the same enzymes (courtesy of Dr. E. Moran, Cold the [3SS]methionine labeling, except for the following modifications. The Spring Harbor Laboratory, NY). The plasmid, carrying the complete adenovi- washings were carried out with 150 mM NaCI-10 mM Tris-HCl, pH 7.20, and rus 5 12S EIA cDNA, was linearized with EcoRI and used in a preparative in the cells were incubated in phosphate-free medium plus 5% dialyzed fetal calf vitro transcription system (Promega). Generally, 30-50 /xg of RNA were serum (Gibco) and 2 mM glutamine. To each flask 12.33 MBq (333 /xCi) of synthesized in a 50-/xl reaction using 100 units of SP6 polymerase. RNA was [32p]orthophosphoric acid (New England Nuclear) were added. Cells were then purified, ethanol precipitated, and stored at -20~ in distilled water. In vitro lysed in 20 mM NaCI-100 mM NaF-30 mM sodium pyrophosphate-3 mM sodium translation was performed using 1 /xg of RNA/50-/xl reaction for 60 min at o-vanadate-50 mM Tris-HCl, pH 8.00-10 mM EDTA-I% Nonidet P-40-1 mM 30~ using a pretreated rabbit reticulocyte lysate (Promega). Translations to PMSF-1 /xg/ml aprotinin-1 /xg/ml leupeptin. generate radioactively labeled protein were done adding to the reaction 37/~Ci (1.37 MBq) of [35S]methionine (Amersham SJ 235) and a minus-methionine Immunoprecipitation amino acid mixture. Translation reactions were stored at -80~

Cell lysates were precleared for 60 min in a rotating device at 4~ by the Coimmunoprecipitations addition of 100/xl/ml of protein A-Sepharose CL-4B (Pharmacia) 25% packed in lysis buffer, conjugated with affinity-purified rabbit anti-mouse IgGs (Cap- Coimmunoprecipitations were always done in a buffer containing no anionic pel). Precleared lysates were then incubated for 180-240 min in a rotating detergents. device at 4~ with 25/xl/ml of protein A-Sepharose 25% packed in lysis buffer, Copreeipitation of pRb with IVT-E1A by the M73 Anti-E1A Antibody. conjugated first with rabbit anti-mouse purified IgGs (Cappel) and then with Precleared 35S-labeled cell lysates and unlabeled in vitro translated E1A (IVT- one of the following antibodies: 1 /xg of purified C36 anti-pRb monoclonal E1A) were incubated for 60 min in a rotating device at 4~ Immunoprecipi- antibody (24) (Oncogene Science); 1 /xg of purified PAb416 anti-SV40 large tation was then carried out with the M73 anti-E1A monoclonal antibody, as T monoclonal antibody (43); 1/xg of purified PMG3-245 anti-pRb monoclonal described above. Twenty ml of IVT-EIA were added for each ml of cell lysate. antibody (27) (PharMingen); 1 /xl of ascites of 1F8 anti-pRb monoclonal Samples were analyzed by SDS-PAGE followed by autoradiography, as de- antibody (44) (courtesy of Dr. D. P. Lane, Medical Science Institute, The scribed above. University of Dundee, Dundee, Scotland); 35 /xl of supernatant of M73 anti- Coprecipitation of IVT-EIA with pRb by the C36 Anti-pRb Antibody. E1A monoclonal antibody (45) (courtesy of Dr. E. Harlow, Cold Spring Harbor Precleared unlabeled lysates from 1 • l06 cells for each cell line (1 ml) and 10 ~1 of 35S-labeled IVT-E1A (~8 x 104 cpm of M73-precipitable radioac- Laboratory, Cold Spring Harbor, NY). After incubation, protein A-Sepharose beads were washed five times at 4~ in lysis buffer. tivity) were incubated for 60 rain in a rotating device at 4~ Immunoprecipi- tation was then carried out using the C36 anti-pRb monoclonal antibody, as SDS-PAGE, Staining, and Fluorography described above. Samples were analyzed by SDS-PAGE followed by autoradiography. When immunoprecipitated samples were analyzed by liquid scin- Thirty-five /xl of electrophoresis sample buffer (62.5 mM Tris-HCl, pH tillation counting, 2 • 106 cells were used. 6.80-2% SDS-5% /3-mercaptoethanol-10% glycerol) was added to drained protein A-Sepharose beads from the last step. Samples were heated at 95~ for Analysis of the Rb mRNA 5 rain and spun to separate protein A-Sepharose beads, and the supernatants were loaded onto a 7.5% or 10% acrylamide gel (46). After the bromophenol The human Rb cDNA sequence used as a reference throughout the present blue tracking dye reached the resolving portion, the gel was run overnight at paper is that published by Friend et al. (51) (GenBank No. M33647). 40 V (constant voltage), stained with Coomassie blue and, in the event of One/xg of total RNA was reverse transcribed using random hexanucleotides labeling with [35S]methionine, treated for fluorography with Amplify (Amer- as primers in the GeneAmp RNA PCR kit (Perkin Elmer Cetus), according to sham) according to the manufacturer's recommendations, then dried and ex- the manufacturer's instructions. One-tenth of the reaction mixture was then used for PCR amplification, using four pairs of oligonucleotide primers, syn- posed at -70~ using Kodak XAR-5 film with Du Pont Cronex intensifying screens. thesized following Horowitz et al. (22). After 40 cycles of amplification, the DNA fragments were analyzed by electrophoresis through 1% agarose gels. Southern Blot Analysis The DGGE analysis was done with a DGGE 2000 system (C.B.S. Scientific Company) (52). Samples were analyzed on perpendicular denaturing gradient Genomic DNA was prepared according to the method of Sambrook et al. gels, done in 1-mm-thick 6.5% polyacrylamide gels containing a 0-80% de- (47), cleaved with HindlII, and resolved by electrophoresis through a 0.8% naturant concentration gradient (5.6 M urea and 32% deionized formamide) agarose gel; 10 ~g of digested DNA were loaded onto each lane. After transfer perpendicular to the direction of the electrophoresis. The gels were run at 60 ~ to nitrocellulose filters (BA-85; Schleicher & Schuell), hybridization was C at 150 V for 6 h in 1 • 800 mM Tris base-400 mM sodium acetate-20 mM carried out with a human Rb cDNA probe (24) labeled with the random EDTA buffer, pH 8.0. For each fragment of Rb to be analyzed, both reference 1099

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1994 American Association for Cancer Research. ABERRANT pRb IN A HUMAN MALIGNANT GLIOMA and sample cDNA were loaded onto the same gel and the separation pattern C'4 was visualized by silver staining (Bio-Rad). < Two different preparations of the cDNA fragment containing the 3' end of ! t/~ t.t. the coding sequence (nt 2275-nt 3046) were made blunt by treatment with T4 t~. tw r'~ polymerase and cloned in a BlueScript SK plasmid linearized by SmaI. The inserted cDNAs were then sequenced using an internal primer, spanning from nt 2847 to nt 2831. kDa RESULTS 97.4 _> pRb Binding with EIA or IVT-E1A. Control experiments for the binding of pRb with adenovirus E1A protein were carried out using 66 _> the 293 cell line, derived from human embryonic cells transformed by a fragment of the adenovirus 5 genome (42) and constitutively expressing the E1A oncoprotein (24). The autoradiogram in Fig. 1, Lane B, shows the immunoprecipitation, after SDS-PAGE, of E1A from 45 _> 35S-labeled 293 cells by the M73 anti-E1A monoclonal antibody (45). IVT-EIA As demonstrated previously (24), E1A appears as various isoforms, ranging from 45 to 55 kDa of apparent molecular mass. Other proteins, such as pRb, p107, p130, and p300, are evident because of their 31_> coimmunoprecipitation with E1A. The same 35S-labeled lysate from 293 cells was immunoprecipitated using the C36 anti-pRb monoclonal antibody (Fig. 1, Lane A). The in vitro translated E1A protein was used to study the interaction 21.5 _> between pRb and E1A. The translation product of the 12S E1A mRNA is a 243-amino acid polypeptide, able to immortalize (53) and trans- 14.5 -> form cultured cells when coexpressed with the adenovirus E1B protein or with an activated Ha-ras cellular oncogene (54, 55). Radiola- Fig. 2. SDS-PAGE (10%) after coimmunoprecipitation of 35S-labeled IVT-EIA pre- beled IVT-E1A, compared with the E1A protein from 293 cells, incubated with LI, DF, CRS-A2, and ADF unlabeled cell lysates; subsequent immuno- displayed a pattern of bands of smaller apparent molecular mass; two precipitation was done using the C36 antibody Left ordinate, molecular weight marker of these, of approximately 38 and 43 kDa, appeared predominant (Fig. migration distances. Autoradiography exposure time was 24 h. 1, Lane C) and both species could be fully recognizable by the M73 monoclonal antibody (Fig. 1, Lane D). Moreover, IVT-E1A was able to interact with pRb. In fact, using the M73 anti-E1A monoclonal with IVT-E1A. This finding is in agreement with the results of Whyte antibody, nonradiolabeled IVT-E1A, added to 35S-labeled lysate from et al. (24), describing the same pattern for pRb in 293 cells immuno- the malignant glioma LI cell line, coimmunoprecipitated with pRb and precipitated with M73, thus confirming the higher affinity of EIA (and with another protein with a higher molecular mass, probably identi- IVT-E1A) for the less phosphorylated forms of the Rb protein. As a fiable as p300 (Fig. 1, Lane E). It should be pointed out, however, that comparison, full pRb electrophoretic polymorphism is shown in Fig. mainly unphosphorylated or underphosphorylated forms (bands with 1, Lane F, where the same LI 35S-labeled lysate was immunoprecipi- faster electrophoretic mobility) of pRb were coimmunoprecipitated tated using the C36 anti-pRb monoclonal antibody. IVT-E1A Binding with pRb from Malignant Glioma Cell Lines. 35S-Labeled IVT-E1A was incubated with unlabeled cell lysates from A B C D E F four different human malignant gliomas, using as few as 1 • 10 6 cells kDa for each cell line. The mixture was then immunoprecipitated choosing 200 _> the C36 anti-pRb monoclonal antibody, as proposed by Whyte et aL (24), because of its ability to recognize the protein even when com- 116._> ] pRb plexed with viral oncoproteins; the proteins were then analyzed by 97.4 _> SDS-PAGE and subsequent autoradiography. Fig. 2 shows the coimmunoprecipitation of 35S-labeled IVT-E1A with pRb in LI, DF, and 66 _.> ADF cell lines and the absence of coimmunoprecipitation in the CRS-A2 cell line. In addition, Table 1 shows that the coimmunopre- EIA 45 ._> cipitated samples from 2 • 106 cells contained specific radioactivity IVT-EIA[ sufficient for liquid scintillation counting. SAOS-2 cells, expressing a truncated Rb gene product unable to bind with E1A (56), were used as 31.-> a negative control. The fact that the only radioactive signal, according to Fig. 2, was the coimmunoprecipitated 35S-labeled IVT-E1A, permitted abolishing SDS-PAGE analysis when a sufficient number of Fig. 1. SDS-PAGE(7.5%) after immunoprecipitationof pRb (LaneA) and E1A (Lane cells was available. B) from 35S-labeled 293 cells by means of the C36 and M73 monoclonal antibodies, Southern and Northern Blot Analysis. Southern blot analysis of respectively. In Lane B, coimmunoprecipitationof several bands, representingpRb, p107, p130, and p300, is evident.Lane C, 35S-labeled IVT-E1A;Lane D, M73-immunoprecipi- DNA from the LI, DF, CRS-A2, and ADF cell lines, after HindlII tated 35S-labeled IVT-E1A; Lane E, unlabeled IVT-E1Aimmunoprecipitation using M73 digestion and probing with Rb cDNA, displayed molecular sizes in 35S-labeled LI malignant glioma cell lysate; coimmunoprecipitation of bands comi- consistent with data reported by other investigators (17) (Fig. 3A). grating with pRb is evident from a comparison with Lane F, where the same lysate is immunoprecipitated using C36. Left ordinate, molecular weight marker migration Moreover, no differences in band and intensity patterns were observed distances. in the four cell lines. 1100

Table 1 Immunoprecipitation and coimmunoprecipitation of radiolabeled IVT-E1A are shown in Fig. 5, where radiolabeled IVT-EIA, coimmunoprecipi- evaluated by means of liquid scintillation counting tated using the C36 antibody, is evident only in LI cells; the other cell Each value is the average +_ SD of two experiments, performed in duplicate, using 2 • 106 cells for each point; 150 _+ 23 cpm should be subtracted from each value, lines failed to display any signal related to radiolabeled IVT-E1A. As representing the nonspecific binding of radiolabeled IVT-E1A with protein A-Sepharose a further control, a nonrelated monoclonal antibody, PAb416, against beads. SV40 large T-antigen (43), was used in LI cells. RadiolabeledIVT-EIA Immunoprecipitated Analysis of the Rb mRNA. Complementary DNA obtained from added to with cpm RNA extracted from the CRS-A2 and 293 cell lines was amplified by Lysis buffer M73 79,650 _ 8,553 PCR using four oligonucleotide pairs, as described in "Materials and LI ceils lysate C36 5,948 -'- 1,405 DF cell lysate C36 3,771 ___ 831 Methods." The four amplified fragments from CRS-A2 cells, when CRS-A2 cell lysate C36 211 • 36 compared by agarose gel electrophoresis, displayed the same size of ADF cell lysate C36 4,415 +_ 675 those obtained from 293 cells (data not shown). This result excluded SAOS-2 cell lysate C36 279 _+ 12 the possibility of large internal deletion of the coding sequence, sug- gesting the presence of a frame-shift mutation on the 3' end. Com- In Northern blot analysis using Rb cDNA as a probe, all the cell parison by DGGE of the PCR fragments coding for the pRb COOH- lines displayed the presence of the message for the Rb gene product, terminal region (nt 2275-nt 3046) from CRS-A2 and 293 cells with a molecular size of 4.7 kilobases (Fig. 3B, top), consistent with revealed the presence of a mutation (data not shown). This fragment data from other laboratories (15, 57). Despite a lower amount of Rb from CRS-A2 cells was then cloned and sequenced, revealing a G message present in the CRS-A2 cell line, no important qualitative insertion at position 2550. This base insertion can lead to the forma- differences were evident among the four malignant glioma cell lines. tion of a stop codon at the level of amino acid 853, producing a Normalization using the glyceroldehyde-3-phosphate dehydrogenase cDNA probe permits comparing the amount of RNA loaded for each O4 single lane (Fig. 3B, bottom). A < Analysis of pRb by Immunoprecipitation. 3SS-Labeled malignant glioma cell lysates were immunoprecipitated using the C36 U= kb anti-pRb monoclonal antibody; lysis and immunoprecipitation were o < carried out in a buffer containing no anionic detergents, such as SDS 9.4._> and DOC. Fig. 4A is an autoradiogram after SDS-PAGE, showing the standard pRb pattern in the LI, DF, and ADF cell lines. In fact, 6.6__.> the protein was found in multiple forms between 105 and 114 kDa of apparent molecular mass; this microheterogeneity is essentially due to different degrees of phosphorylation and is considered normal 4.3__._> for cycling, nonsynchronized cells (4-6, 24). CRS-A2 cells, instead, showed a band with a significatively lower apparent molecular mass, of about 99 kDa, with no discernible microheterogeneity pattern (Fig. 4A). To establish whether this abnormal band was an artifact, immunoprecipitation was done again using two more, highly spe- 2.3___> cific, anti-pRb monoclonal antibodies, such as 1F8 (44) and PMG3- 2.0__> 245 (27). The same immunoprecipitation pattern was observed with both antibodies, as with the C36 antibody, where immunoprecipitation was carried out either in the absence or in the presence of SDS and DOC (Fig. 4B). As a comparison, the standard pRb immunoprecipitation pattern by C36 is represented by the LI cell line. The 99- kDa CRS-A2 aberrant Rb protein was therefore recognized in immu- < noprecipitation by three different specific monoclonal antibodies against the human Rb protein and, in the case of C36, in the B ~ o 0 < presence of anionic detergents. The ability of the CRS-A2 aberrant protein to become phosphory- 28S _.> lated was determined by immunoprecipitation after cell labeling with Rb [3ep]-orthophosphoric acid, using the same C36 antibody. As shown in Fig. 4C, phosphorylated pRb (ppRb) was clearly present in LI cells, but the same procedure failed to detect any phosphorylation signal 18S _> corresponding to the migrating distance of the CRS-A2 Rb gene ! product. IVT-E1A Binding with pRb from Other Malignant Cell Lines. To investigate the ability to recognize pRb abnormalities other than GA-P-DH that reported for CRS-A2, the assay was extended to some other human tumor cell lines bearing described pRb deficiencies. Therefore Fig. 3. A, Southern blot analysis, using a human Rb cDNA probe, of genomic DNA IVT-E1A coimmunoprecipitation assay was done on two cell lines from LI, DF, CRS-A2, and ADF malignant gliomas after HindIIl digestion and electrophoresis through 0.8% agarose. Kilobase pairs (kb) are molecular size markers with their bearing truncated forms of pRb, such as SAOS-2 osteosarcoma cell respective values. A, 1.8-kilobase Rb DNA fragment is visible only upon longer exposure. line (58) and Y79 (38, 59); on WERI-Rbl retinoblastoma cell line, B, Top: Northern blot analysis, using a human Rb cDNA probe, of total RNA from LI, DF, with a deletion of the Rb gene (39); and on the H209 small cell lung CRS-A2, and ADF malignant gliomas after electrophoresis through 1.2% agarose gel in the presence of formaldehyde. Arrows, migrating distances of 28S and 18S rRNA. Bottom, cancer cell line, bearing a point mutation leading to the substitution of the same nitrocellulose reprobed with glyceraldehyde-3-phosphate dehydrogenase (GA- a single amino acid in the pRb molecule (Cys 706 to Phe) (40). Results P-DH) cDNA. 1101 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1994 American Association for Cancer Research. ABERRANT pRb IN A HUMAN MALIGNANT GLIOMA

CRS-A2 LI

O4 O4 < B .,(

A ' Ab ~ ~,- U'~ J, o C < SDS.DOC - - - + - kOo kDa 200 _._> .... - 9 200 _> i"! 200 ._>

116_> ~ " pRb <._ ppRb ] pRb 97.4 --> 97.4 _~ ~,~'~'., 97.4 ~ ~ ...... 69 ._> 66 ._> ~i~,. 66 _> ~

9 ~, 46 ._>

45 _.> i 30

Fig. 4. A, 7.5% SDS-PAGE after immunoprecipitation in 35S-labeled LI, DF, CRS-A2, and ADF malignant glioma cell lysates using the C36 anti-pRb monoclonal antibody (Ab). B, 7.5% SDS-PAGE after immunoprecipitation in 3SS-labeledCRS-A2 malignantglioma cell lysate using C36, IF8, PMG3-245, and C36 again, but in the presence of SDS and DOC (See "Materials and Methods"). As a comparison, 35S-labeled LI cell lysate immunoprecipitated using C36 is reported. C, 7.5% SDS-PAGE after immunoprecipitation in 32p-labeled LI and CRS-A2 malignant glioma cell lysates using C36; Rb protein is indicated as ppRb. Left ordinate, molecular weight marker migration distances.

truncated Rb protein, lacking 75 amino acids. The experiment was nary screening (see Table 1), since any radioactive signal could be done twice, starting from two different RNA preparations. related only to the presence of coimmunoprecipitated IVT-E1A. We attempted to define more accurately the molecular defect im- DISCUSSION pairing the E1A-binding properties in the CRS-A2 Rb gene product. The immunoprecipitation analysis of the CRS-A2 cell lysate using an A functional analysis of the Rb gene in four human malignant anti-pRb antibody showed the presence of a pRb with a lower appar- glioma cell lines was done by an approach based on the coimmuno- ent molecular mass in SDS-PAGE, when compared with normal pRb. precipitation of a complex formed by 35S-labeled IVT-E1A, pRb, and In addition, this protein displayed a low or a null degree of phos- an anti-pRb monoclonal antibody. This allowed us to discover that an phorylation, perhaps due to its peculiar primary structure; the aberrant abnormal Rb gene product, unable to bind with IVT-E1A, was present in the CRS-A2 malignant glioma cells. Other methods, such as South- protein, in fact, might not become an efficient substrate for the spe- ern and Northern blots, did not discriminate between CRS-A2 and the cific kinases or might not be compartmentalized in the nucleus. These other malignant gliomas tested. Some investigators report that certain hypotheses are not mutually exclusive. retinoblastomas express truncated Rb transcripts (60, 61); Venter et al. Binding with E1A takes place in a specific domain of the pRb (19) find in a human malignant glioma a mRNA species with a smaller molecule, functionally designed as "pocket," spanning from amino transcript which, however, seemed to be absent in all our cultured acid 379 to 793, which is the minimal region necessary for EIA malignant glioma cell lines. Only more complex techniques, such as binding (62). Molecular damages at the level of the pocket region can immunoprecipitation performed on total labeled cellular proteins or lead to pRb malfunction in binding viral oncoproteins as well as DNA sequencing, were able to detect an alteration in the CRS-A2 Rb endogenous transcriptional factors (22, 40, 41, 44, 63). The analysis of gene product and to clarify its nature. the CRS-A2 Rb cDNA sequence, revealing the presence of a G in- The method of radiolabeled IVT-E1A coimmunoprecipitation by sertion at nt 2550, was consistent with our previous results. This the C36 monoclonal antibody, followed by SDS-PAGE and autoradi- insertion can lead, in fact, to the generation of a COOH-terminally ography, was quite sensitive, because as few as 1 • 10 6 cultured cells truncated pRb, lacking the last 75 amino acids, with a computed were used for each single assay (see Fig. 2). Prolongation of the molecular mass of 97,663 Da. The molecular damage found in autoradiography exposure time beyond 24 h enhanced the sensitivity CRS-A2 pRb could be the cause of the lack of interaction with of the method, thus allowing a decrease of both the cell number and IVT-E1A and of the lack of phosphorylation as well. Unless there are the amount of radiolabeled IVT-EIA (data not shown). Furthermore, sense point mutations in the unsequenced upstream region, leading to it should be emphasized that the functional aspect of the assay per- the substitution of critical amino acids, these data suggest that the mitted detecting different kinds of Rb gene product abnormalities, 75-amino acid COOH-terminal region may be necessary for the E1A leading to defective binding with IVT-E1A (see Fig. 5). binding. In addition, a simple liquid scintillation counting after coimmuno- In addition, the CRS-A2 Rb gene product was detected in immu- precipitation, instead of the more complex and time-consuming SDS- noprecipitation by the 1F8 monoclonal antibody, which does not rec- PAGE and autoradiography, might be sufficient for a rapid, prelimi- ognize SAOS-2 pRb. The defective Rb protein present in the SAOS-2 1102

and M. Di Giovanni for the photographic work and to Professor L. Pozzi for critically reviewing the manuscript.

REFERENCES = =,,, >. 1. Klein, G. The approaching era of the tumor suppressor genes. Science (Washington KDa DC), 238: 1539-1545, 1987. 2. Hansen, M. K, and Cavenee, W. K. Retinoblastoma and the progression of tumor genetics. Trends Genet., 4: 125-128, 1988. 97.4 ..> 3. Weinberg, R. A. The retinoblastoma gene and cell growth control. Trends Biochem. Sci., 15: 199-202, 1990. 66 ..> 4. Buchkovich, K., Duffy, L. A., and Harlow, E. The retinoblastoma protein is phosphorylated during specific phases of the cell cycle. Cell, 58: 1097-1105, 1989. 5. Chen, P. L., Scully, P., Shew, J. Y., Wang, J. Y., and Lee, W. H. Phosphorylation of the retinoblastoma gene product is modulated during the cell cycle and cellular differ- 45 -> entiation. Cell, 58: 1193-1198, 1989. 6. DeCaprio, J. A., Ludlow, J. W., Lynch, D., Furukawa, Y., Griffin, J., Piwnica Worms, IVT-EIA H., Huang, C. M., and Livingston, D. M. The product of the retinoblastoma susceptibility gene has properties of a cell cycle regulatory element. Cell, 58: 1085-1095, 1989. 31 7. Dryja, T. P., Rapaport, J. M., Joyce, J. M., and Petersen, R. A. Molecular detection of -> deletions involving band q14 of chromosome 13 in retinoblastomas. Proc. Natl. Acad. Sci. USA, 83: 7391-7394, 1986. 8. Knudson, A. G., Jr. Hereditary cancers disclose a class of cancer genes. Cancer (Phila.), 63: 1888-1891, 1989. 21.5 ..> 9. Harbour, J. W., Lai, S. L., Whang Peng, J., Gazdar, A. E, Minna, J. D., and Kaye, E J. Abnormalities in structure and expression of the human retinoblastoma gene in SCLC. Science (Washington DC), 241: 353-357, 1988. 14.5 ._> 10. Hensel, C. H., Hsieh, C. L., Gazdar, A. F., Johnson, B. E., Sakaguchi, A. Y., Naylor, S. L., Lee, W. H., and Lee, E. Y. Altered structure and expression of the human retinoblastoma susceptibility gene in small cell lung cancer. Cancer Res., 50: 3067-

m 3072, 1990. 11. T'Ang, A., Varley, J. M., Chakraborty, S., Murphree, A. L., and Fung, Y. K. Structural Ab PAb C36 rearrangement of the retinoblastoma gene in human breast carcinoma. Science (Wash- 416 ington DC), 242: 263-266, 1988. Fig. 5. SDS-PAGE (10%) after coimmunoprecipitation of 35S-labeled IVT-EIA prein- 12. Varley, J. M., Armour, J., Swallow, J. E., Jeffreys, A. J., Ponder, B. A., T'Ang, A., cubated with LI, SAOS-2, Y79, WERI-Rbl, and H209 unlabeled cell lysates; subsequent Fung, Y. K., Brammar, W. J., and Walker, R. A. The retinoblastoma gene is frequently immunoprecipitation was done for all the samples using the C36 antibody; as a control, altered leading to loss of expression in primary breast tumours. Oncogene, 4: 725- also the PAb416 antibody was used for the LI cells. Left ordinate, molecular weight 729, 1989. marker migration distances. Autoradiography exposure time was 24 h. 13. Horowitz, J. M., Park, S. H., Yandell, D. W., and Weinberg, R. A. Involvement of the retinoblastoma gene in the genesis of various human tumors. In: W. Cavenee, N. Hastie, and E. Stanbridge (eds.), Recessive Oncogenes and Tumor Suppression, pp. 101-107. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 1989. osteogenic sarcoma cell line is a 95-kDa protein that lacks exons 14. Hansen, M. E Loss of the retinoblastoma susceptibility gene is heterogeneous in 21-27. It is unable to bind with SV40 large T and E1A oncoproteins, osteosarcomas. In: W. Cavenee, N. Hastie, and E. Stanbridge (eds.), Recessive On- cogene and Tumor Suppression, pp. 125-131. Cold Spring Harbor, NY: Cold Spring is found only in an underphosphorylated form and is recognized in the Harbor Laboratory, 1989. cytoplasm by immunofluorescence using the PMG3-245 monoclonal 15. Furukawa, Y., DeCaprio, J. A., Belvin, M., and Griffin, J. D. Heterogeneous expres- antibody, but not the 1F8 (41, 44, 56). The epitope of the 1F8 antibody sion of the product of the retinoblastoma susceptibility gene in primary human leukemia cells. Oncogene, 6: 1343-1346, 1991. is supposed to lie within the pRb segment encoded by exons 21-27, 16. Bookstein, R., Shew, J. Y., Chen, P. L., Scully, P., and Lee, W. H. Suppression of which is absent in the SAOS-2 pRb (44). The ability of 1F8 antibody tumorigenicity of human prostate carcinoma cells by replacing a mutated RB gene. to recognize the CRS-A2 Rb gene product may indicate that the DNA Science (Washington DC), 247: 712-715, 1990. 17. Horowitz, J. M., Park, S. H., Bogenmann, E., Cheng, J. C., Yandell, D. W., Kaye, F. sequence encoding for its epitope is between nt 2110 (the beginning J., Minna, J. D., Dryja, T. P., and Weinberg, R. A. Frequent inactivation of the of exon 21) and nt 2550. retinoblastoma anti-oncogene is restricted to a subset of human tumor cells. Proc. Natl. Acad. Sci. USA, 87: 2775-2779, 1990. The procedure of the IVT-E1A coimmunoprecipitation with pRb, 18. Bookstein, R., Rio, P., Madreperia, S. A., Hong, F., Allred, C., Grizzle, W. E., and Lee, described in the present study, is therefore proposed as a quick, easy W. H. Promoter deletion and loss of retinoblastoma gene expression in human prostate to use, and reproducible method for screening the functional state of carcinoma. Proc. Natl. Acad. Sci. USA, 87: 7762-7766, 1990. 19. Venter, D. J., Bevan, K. L., Ludwig, R. L., Riley, T. E., Jat, P. S., Thomas, D. G., and the Rb protein, based on its binding properties with the E1A onco- Noble, M. D. Retinoblastoma gene deletions in human glioblastomas. Oncogene, 6: protein. Since endogenous transcription factors, such as E2F, share the 445-448, 1991. same binding site on the oncosuppressor gene product with E1A (30), 20. Godbout, R., Miyakoshi, J., Dobler, K. D., Andison, R., Matsuo, K., Allalunis Turner, M. J., Takebe, H., and Day, R. S. Lack of expression of tumor-suppressor genes in the IVT-E1A binding assay could be considered as a functional test for human malignant glioma cell lines. Oncogene, 7: 1879-1884, 1992. identifying of malfunctions in the pRb molecule. 21. Lee, W. H., Shew, J. Y., Hong, E D., Sery, T. W., Donoso, L. A., Young, L. J., Bookstein, R., and Lee, E. Y. The retinoblastoma susceptibility gene encodes a nuclear Molecular cloning of the p107, a pRb-like molecule, shows signifi- phosphoprotein associated with DNA binding activity. Nature (Lond.), 329: 642-645, cant homology between this protein and the pRb in the pocket 1987. region (64). Because also p107 binds with E1A (24, 64) and the 22. Horowitz, J. M., Yandell, D. W., Park, S. H., Canning, S., Whyte, P., Buchkovich, K., Harlow E., Weinberg, R. A., and Dryja, T. P. Point mutational inactivation of the cellular factor E2F as well (30-32), a similar approach for the func- retinoblastoma antioncogene. Science (Washington DC), 243: 937-940, 1989. tional analysis of p107 or other E1A-binding molecules could be 23. Mihara, K., Cao, X. R., Yen, A., Chandler, S., Driscoll, B., Murphree, A. L., T'Ang, investigated. A., and Fung, Y. K. Cell cycle-dependent regulation of phosphorylation of the human retinoblastoma gene product. Science (Washington DC), 246: 1300-1303, 1989. 24. Whyte, P., Buchkovich, K. J., Horowitz, J. M., Friend, S. H., Raybuck, M., Weinberg, R. A., and Harlow, E. Association between an oncogene and an anti-oncogene: the ACKNOWLEDGMENTS adenovirus E1A proteins bind to the retinoblastoma gene product. Nature (Lond.), 334: 124-129, 1988. The authors thank Dr. G. Zupi for providing the DF cell line, Dr. W. Malorni 25. Huang, H. J., Yee, J. K., Shew, J. Y., Chen, P. L., Bookstein, R., Friedmann, T., Lee, for the ADF cell line, Dr. M. Zamanian for the SAOS-2 cell line, Dr. A. Albini E. Y., and Lee, W. H. Suppression of the neoplastic phenotype by replacement of the RB gene in human cancer cells. Science (Washington DC), 242: 1563-1566, 1988. for the Y79 and WERI-Rbl cells, Dr. D. P. Lane for the 1F8 anti-pRb mono- 26. Takahashi, R., Hashimoto, T., Xu, H. J., Hu, S. X., Matsui, T., Miki, T., Bigo Marshall, clonal antibody, Dr. E. Harlow for the M73 anti-E1A monoclonal antibody, and H., Aaronson, S. A., and Benedict, W. E The retinoblastoma gene functions as a Dr. G. D'Agostino for oligonucleotide synthesis and are grateful to I. Zardin growth and tumor suppressor in human bladder carcinoma cells. Proc. Natl. Acad. Sci. 1103

USA, 88: 5257-5261, 1991. 45. Harlow, E., Franza, B. R., Jr., and Schley, C. Monoclonal antibodies specific for 27. DeCaprio, J. A., Ludlow, J. W., Figge, J., Shew, J. Y., Huang, C. M., Lee, W. H., adenovirus early region 1A proteins: extensive heterogeneity in early region 1A Marsilio, E., Paucha, E., and Livingston, D. M. SV40 large tumor antigen forms a products. J. Virol., 55: 533-546, 1985. specific complex with the product of the retinoblastoma susceptibility gene. Cell, 54: 46. Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of the 275-283, 1988. bacteriophage T4. Nature (Lond.), 227." 680-685, 1970. 28. Ludlow, J. W., DeCaprio, J. A., Huang, C. M., Lee, W. H., Paucha, E., and Livingston, 47. Sambrook. J., Fritsch, E. E, and Maniatis, T. Molecular Cloning: a Laboratory D. M. SV40 large T antigen binds preferentially to an underphosphorylated member Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 1989. of the retinoblastoma susceptibility gene product family. Cell, 56: 57-65, 1989. 48. Feinberg, A. E, and Vogelstein, B. A technique for radiolabeling DNA restriction 29. Dyson, N., Howley, E M., Munger, K., and Harlow, E. The human papilloma virus-16 endonuclease tYagments to high specific activity. Anal. Biochem., 132: 6-13, 1983. E7 oncoprotein is able to bind to the retinoblastoma gene product. Science (Wash- 49. Chomczynski, E, and Sacchi, N. Single-step method of RNA isolation by acid ington DC), 243: 934-937, 1989. guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem., 162: 156- 30. Chellappan, S., Kraus, V. B., Kroger, B., Munger, K., Howley, P. M., Phelps, W. C., 159, 1987. and Nevins, J. R. Adenovirus E1A, simian virus 40 tumor antigen, and human 50. Zerler, B,, Moran, B., Maruyama, K., Moomaw, J., Grodzicker, T., and Ruley, H. E. papillomavirus E7 protein share the capacity to disrupt the interaction between tran- Adenovirus E IA coding sequences that enable ras and pint oncogenes to transform scription factor E2F and the retinoblastoma gene product. Proc. Natl. Acad. Sci. USA, cultured primary cells. Mol. Cell. Biol., 6: 88%899, 1986. 89: 4549--4553, 1992. 51. Friend, S. H., Horowitz, J. M., Gerber, M. R., Wang, X. E, Bogenmann, E., Li, E E, 31. Cao, L., Faha, B., Dembski, M., Tsai, L. H., Harlow, E., and Dyson, N. Independent and Weinberg, R. A. Deletions of a DNA sequence in retinoblastomas and mesen- binding of the retinoblastoma protein and p107 to the transcription factor E2E Nature chymal tumors: organization of the sequence and its encoded protein. Proc. Natl. (Lond.), 355: 176-179, 1992. Acad. Sci. USA, 84: 9059-91163, 1987. 32. Shirodkar, S., Ewen, M., DeCaprio, J. A., Morgan, J., Livingston, D. M., and 52. Myers, R. M., Maniatis, T., and Lerman, L. S. Detection and localisation of single Chittenden, T. The transcription factor E2F interacts with the retinoblastoma product base change by denaturing gradient gel electrophoresis. Methods Enzymol., 155: and a pl07-cyclin A complex in a cell cycle-regulated manner. Cell, 68: 157-166, 501-527, 1987. 1992. 53. Houweling, A., van der Elsen, E J., and van der Eb, A. J. Partial transformation of 33. Imai, Y., Matsushima, Y., Sugimura, T., and Terada, M. Purification and character- primary rat cells by the leftmost 4.5% fragment of adenovirus 5 DNA. Virology, 105: ization of human papillomavirus type 16 E7 protein with preferential binding capacity 537-550, 1980. to the underphosphorylated form of retinoblastoma gene product. J. Virol., 65: 4966- 54. Ruley, H. E. Adenovirus early region 1A enables viral and cellular transforming genes 4972, 1991. to transform primary cells in culture. Nature (Lond.), 304: 602--606, 1983. 34. Nevins, J. R. E2F: a link between the Rb tumor suppressor protein and viral onco- 55. van der Elsen, E, Houweling, A., and van der Eb, A. J. Expression of region E1B of proteins. Science (Washington DC), 258: 424-429, 1992. human adenovirus in the absence of region E1A is not sufficient for complete 35. Paggi, M. G., Zupi, G., Fanciulli, M., Del Carlo, C., Giorno, S., Laudonio, N., transformation. Virology, 128: 377-390, 1983. Silvestrini, B., Caputo, A., and Floridi, A. Effect of Ionidamine on the utilization of 56. Templeton, D. J., Park, S. H., Lanier, L., and Weinberg, R. A. Nonfunctional mutants 14C-labeled glucose by human astrocytoma cells. Exp. Mol. Pathol., 47: 154-165, of the retinoblastoma protein are characterized by defects in phosphorylation, viral 1987. oncoprotein association, and nuclear tethering. Proc. Natl. Acad. Sci. USA, 88: 36. Savarese, A., Annicchiarico Petruzzelli, M., Citro, G., Zupi, G., Spagnoli, L. G., 3033-3037, 1991. Colantoni, A., Vernole, P., Stephanou, A., Knight, R. A., and Guerrieri, P. Character- 57. Rubin, S. J., Hallahan, D. E., Ashman, C. R., Brachman, D. G., Beckett, M. A., ization of a human glioblastoma cell line (LI) expressing hypothalamic and pituitary Virudachalam, S., Yandell, D. W., and Weichselbaum, R. R. Two prostate carcinoma hormones. Exp. Brain Res., 89: 408-414, 1992. cell lines demonstrate abnormalities in tumor suppressor genes. J. Surg. Oncol., 46: 37. Zupi, G., Candiloro, A., Laudonio, N., Carapella, C., Benassi, M., Riccio, A., Bel- 31-36, 1991. locci, M., and Greco, C. Establishment, characterization and chemosensitivity of two 58. Shew, J. Y., Lin, B. T., Chert, E L., Tseng, B. Y., Yang Feng, T. L., and Lee, W. H. human glioma derived cell lines. J. Neurooncol., 6: 169-177, 1988. C-terminal truncation of the retinoblastoma gene product leads to functional inacti- 38. Lee, E. Y., Bookstein, R., Young, L. J., Lin, C. J., Rosenfeld, M. G., and Lee, W. H. vation. Proc. Natl. Acad. Sci. USA, 87." 6--10, 1990. Molecular mechanism of retinoblastoma gene inactivation in retinoblastoma cell line 59. Bookstein, R., Lee, E. Y., To, H., Young, L. J., Sery, T. W., Hayes, R. C., Friedmann, Y79. Proc. Natl. Acad. Sci. USA, 85: 6017-6021, 1988. T., and Lee, W. H. Human retinoblastoma susceptibility gene: genomic organization 39. Muncaster, M. M., Cohen, B. L., Phillips, R. A., and Gallie, B. L. Failure of RB1 to and analysis of heterozygous intragenic deletion mutants. Proc. Natl. Acad. Sci. USA, reverse the malignant phenotype of human tumor cell lines. Cancer Res., 52: 654- 85: 2210-2214, 1988. 661, 1992. 60. Fung, Y. K., Murphree, A. L., T'Ang, A., Qian, J., Hinrichs, S. H., and Benedict, W. 40. Bignon, Y. J., Shew, J. Y., Rappolee, D., Naylor, S. L., Lee, E. Y., Schnier, J., and Lee, E Structural evidence for the authenticity of the human retinoblastoma gene. Science W. H. A single Cys706 to Phe substitution in the retinoblastoma protein causes the (Washington DC), 236: 1657-1661, 1987. loss of binding to SV40 T antigen. Cell Growth Differ., 1: 647-651, 1990. 61. Lee, W H., Bookstein, R., Hong, E, Young, L. J., Shew, J. Y., and Lee, E. Y. Human 41. Shew, J-Y., Lin, B. T-Y., Chen, P. L., Tseng, B. Y., Yang-Feng, T. L., and Lee, W. H. retinoblastoma susceptibility gene: cloning, identification, and sequence. Science C-terminal truncation of the retinoblastoma gene product leads to functional inacti- (Washington DC), 235: 1394-1399, 1987. vation. Proc. Natl. Acad. Sci. USA, 87: 6--10, 1990. 62. Kaelin, W. G., Jr., Pallas, D. C., DeCaprio, J. A., Kaye, E J., and Livingston, D. M. 42. Graham, F. L., Smiley, J., Russell, W. C., and Nairn, R. Characteristics of a human cell Identification of cellular proteins that can interact specifically with the T/E1A-binding line transformed by DNA from human adenovirus type 5. J. Gen. Virol., 36: 59-74, region of the retinoblastoma gene product. Cell, 64: 521-532, 1991. 1977. 63. Hu, Q. J., Dyson, N., and Harlow, E. The regions of the retinoblastoma protein needed 43. Harlow, E., Crawford, L. V., Pim, D. C., and Williamson, N. M. Monoclonal anti- for binding to adenovirus EIA or SV40 large T antigen are common sites for bodies specific for simian virus 40 tumor antigens. J. Virol., 39: 861-869, 1981. mutations. EMBO J., 9:1147-1155, 1990. 44. Bartek, J., Vojtesek, B., Grand, R. J., Gallimore, P. H., and Lane, D. P. Cellular 64. Ewen, M. E., Xing, Y. G., Lawrence, J. B., and Livingston, D. M. Molecular cloning, localization and T antigen binding of the retinoblastoma protein. Oncogene, 7: 101- chromosomal mapping, and expression of the cDNA for p107, a retinoblastoma gene 108, 1992. product-related protein. Cell, 66:1155-1164, 1991.

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Marco G. Paggi, Fabio Martelli, Maurizio Fanciulli, et al.

Cancer Res 1994;54:1098-1104.

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