( RESEARCH 51.6.W-6.W. tX-ccmbcrI. I99I|

Advances in Brief and Abnormal in -induced Human Sarcomas1

David G. Brachman,2 Dennis E. Hallaban, Michael A. Beckett, David W. Yandell, and Ralph R. Weichselbaum

Department of Radiation and Cellular , University of Chicago, Chicago, Illinois 60637 [D. G. B., D. E. H., M. A. B., R. R. W.], and Department of Ophthalmology, Massachusetts Eye and Ear Infirmary, and Harvard Medical School, Boston, Massachusetts 02114 ¡D.W. Y.J

Abstract a 4.7-kilobase mRNA transcript is expressed in most normal tissues. The gene encodes a nuclear phosphoprotein that com The potentially carcinogenic effect of therapeutic irradiation has been plexes with transforming of the adeno- and papovavi- recognized for many years. Second malignancies, usually , are known to arise within or at the edge of radiation fields after a period of rus types in in vitro assays (12, 13). It is likely that the ability several years after the initial radiation exposure. We analyzed tumor of the -encoded oncoproteins to form complexes with the cells derived from seven radiation-induced tumors for abnormalities in Rb protein is tightly linked to their transforming potential (14). tumor suppressor p53 and retinoblastoma at the DNA sequence The Rb gene has the properties of a , and/or protein level. p53 mutations were detected by exon-specific polym- inasmuch as loss of both copies of the gene favors malignant erase chain reaction amplification and single-strand conformation poly transformation. This model was confirmed by the isolation and morphism analysis of exons 5-8 followed by direct genomic sequencing characterization of a gene demonstrating the expected proper of those tumors exhibiting a variant pattern. The p53 gene was abnormal ties of the Rb , including suppression of the malignant in three of six sarcomas studied. Retinoblastoma gene analysis was performed by Western immunoblot; was under- phenotype in some retinoblastoma and cells in vitro (15, 16). In addition to and sarcomas, the phosphorylated in three of seven tumors and absent in one other. In all, six of seven radiation-induced human tumors have abnormalities of one Rb gene if found in mutated form in a number of human or both suppressor genes. Inactivation of tumor suppressor genes by malignancies, including small (17, 18) and may contribute to radiation . bladder (19), breast (20), and prostate (21, 22). p53 is a nuclear phosphoprotein encoded by a gene on the Introduction short arm of 17. In addition to sarcomas (9), 17p allelic losses have been demonstrated in a variety of human The occurrence of new solid tumors is well-recognized but tumors including colon (7), breast (7), brain (7), esophagus (23), infrequent consequence of therapeutic radiation (1, 2). RITs' hepatomas (24), bladder (25), and prostate (22); germinal p53 are often sarcomas, and they share several characteristics, in mutations are present in patients with the Li-Fraumeni syn cluding a relatively long latency period after initial radiation drome (26, 27). Often, onep5J alíeleisdeleted while the second (3-30 years), a location at or within the margins of previous alíeleis found to be mutated. Although the exact function of irradiation, and a histology different from that of the original thep5J gene remains unknown, wild-type p53 is a DNA-binding tumor. Although ionizing radiation was one of the first proven protein and has some of the properties of a transcriptional environmental (3, 4), the molecular mechanism(s) activator (28, 29). Increased p53 protein stability, altered pro of radiation carcinogenesis remain somewhat obscure. Initial tein confirmation (30), and the ability to form pseudohomodi- studies of radiation carcinogenesis described activation of the mers with 70/wild-type p53 protein have ras family of in experimental systems (5). Recently, been demonstrated as a result ofp53 gene mutations (31). This attention has been focused on the inactivation of tumor sup latter property may be a means by which heterozygous p53 pressor genes in solid tumor carcinogenesis (6). Two such genes, mutations negate the action of wild-type p53 and exert a dom Rb andp5J, have been implicated in the etiology of a variety of inant negative effect (32, 33). Taken together, these studies tumors, including spontaneously occurring sarcomas (7-9). In suggest that the tumor suppressor function of the wild-type p53 addition, patients with hereditary retinoblastoma, who carry protein may be related to transcriptional regulation and appears one defective Rb alíele,have a high incidence of radiation- to be abrogated by allelic loss or replacement with an abnormal induced sarcomas (10, 11). For these reasons, we hypothesized form. that ionizing radiation may play a role in the alteration of these To investigate whether alterations of tumor suppressor genes tumor suppressor genes in irradiated tissue, thereby contribut Rb and p53 are associated with human radiation-induced sar ing to the development of radiation-induced osteo- and soft- comas, we examined tumor cells from seven radiation-induced tissue sarcomas. osteo- and soft-tissue sarcomas at the Rb and p53 loci. For p53, The Rb gene contains 27 exons spanning 200 kilobases, and we chose to examine exons 5-8, since they are evolutionarily conserved regions of p53 and may contain mutational hotspots Received 9/9/91; accepted 10/11/91. The costs of publication of this article were defrayed in part by the payment (7). All tumors were of a histology different from that of the of page charges. This article must therefore be hereby marked advertisement in original tumors, occurred within or at the margin of radiation accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This work was supported by NI Health Grant CA41068. the Chicago Tumor field, and arose 6 to 20 years after therapeutic radiation. Institute, a gift from the Passis Family, and the Center for . 2To whom requests for reprints should be addressed, at Department of Materials and Methods Radiation and Cellular Oncology, University of Chicago, 5841 S. Maryland Avenue, Box 442, Chicago, IL 60637. ' The abbreviations used are: RIT, radiation-induced tumor; PCR. polymerase Cell Culture. The tissue was processed as previously described (34). RIT lb,2,3,5,6, STSAR 5. and STSAR 28 were cultured in Dulbecco's chain reaction: SSCP, single-strand conformation : Rb. retinoblas toma; pRb. retinoblastoma protein: TBS, Tris-buffered saline: BSA, bovine serum modification of Eagle's medium/Ham's nutrient mixture F-12 with albumin. 20% fetal bovine serum and grown to 80% confluence in 150-ml flasks: 6393

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IMR-90, Y-79, and COS cells (American Type Culture Collection) is shown in Fig. 1. In RIT-lb, an 8- were cultured similarly, except for the use of 10% fetal bovine serum. starting at codon 250 of exon 7 resulted in a frame shift and Tumor cells from RIT Ib and STSAR 28 were placed into short-term generation of a premature stop codon near the 5' end of exon culture; cells from the remaining samples became established lines. 8 (data not shown). In STSAR-5, we were unable to amplify SSCP Analysis and Direct Sequencing. SSCP analysis of p53 exons 5-8 was performed using only minor modifications of the procedure any of the fragments within the screened region, suggesting a published by Orila et al. (35). For p53, exon 7, a 204-base pair PCR possible deletion of the p53 gene, which was confirmed on (36) fragment was generated from 50 ng of genomic DNA in a 50-pl Southern analysis (data not shown). mixture containing: 20 pM dATP, dTTP, or dGTP); 2 pM dCTP; 2.5 For the Rb gene, protein analysis was performed by Western mM MgCh; 20 pmol of each primer; 10 mM Tris (pH 8.4); 50 mM KO; immunoblot. Protein was extracted from early (third) passage 50 jig/ml BSA; 0.5 units Taq polymerase (Perkin-Elmer Cetus); and cell lines. Fig. 2 shows a Western immunoblot performed with 0.1 pi of |a-"P]dCTP (3000 Ci/mmol). The primers used were 5'- antibody PMG3-245, which recognizes the phosphorylated GGCCTCATCTTGGGCCTGTG-3' and 5'-CAGTGTGCAGG- form of the retinoblastoma protein (pll2-114Rb) and the GTGGCAAGT-3'. PCR was carried out for 30 cycles (of 30 s at 94°C, 90 s at 60°C,and 120 s at 71°C)using a Programmable Thermal Cycler (Perkin-Elmer/Cetus). One-tenth of the crude amplified product was O ' mixed with 40 pi of a 0.1% sodium dodecyl sulfate-10 mM EDTA A mixture, followed by 1:1 dilution with a 95% formamide-20 mM EDTA- » » a) Control b) Mutant 0.05% bromophenol blue-0.05% xylene cyanol loading solution. Di C luted samples were heat-denatured at 95°Cfor 2 min and then loaded G on 6% nondenaturing polyacrylamide gels and once on a gel containing GATC GATC 10% glycerol. Electrophoresis was carried out at room temperature T with a constant power of 7 W for 8 to 16 h. Autoradiography was A performed for 12-24 h without an intensifying screen. Direct genomic sequencing of double-stranded PCR fragments was performed. Briefly, C a PCR fragment spanning exons 7 and 8 was amplified using the C oligonucleotides 5'-AAAGGCCTCCCCTGCTTGCC-3' and 5'- C TCTCCTCCACCGCTTCTTGT-3'. Amplified DNA samples were pu G rified through Centricon 100 microconcentrators (Amicon) and com bined with 20 pmol of one of several different -"P-end-labeled primers C internal to the amplified fragment. Primer-template mixtures were heat- C denatured, and direct DNA sequencing (37) was carried out using the G Sequenase (U.S. Biochemical). Electrophoresis was on 8% denaturing polyacrylamide gels. Autoradiography was carried out for 12-24 h without an intensifying screen. A Protein Extraction and Western Immunoblots. The pi 10 Rb was extracted as previously described (13). Cells were washed in ice- cold TBS (pH 8.0) and scraped into TBS at 4"C. Cells were centrifuged at 3000 rpm at 4°C.One to 2 ml of cold extraction buffer C (50 mM Tris, pH 8.0, 120 mM NaCl, 0.5% NP40, 100 mM NaF, 200 pM ovanadate, and 10 pg/ml each aprotinin, phenylmethylsulfonyl fluoride, and leupeptin) were added to the cells, and the cells were vortexed. Protein quantification (BioRad) was followed by the addition of 150 pg Fig. I. Sequencing auloradiograph of lhep53 gene in the region of codon 245 for l'< k amplified DNA samples from a normal fibroblast cell line, IMR-90 (a), of total cell lysate to each gel slot and size fractionation (13) on a and a radiation-induced osteosarcoma cell line, RIT-2 (b). a, sequence of normal sodium dodecyl sulfate-polyacrylamide gel (7.5%). Protein was then fibroblast cell line with normal noncoding codon 245 sequence (CCG). *, non- electrotransferred to an Imobilon nylon membrane (Millipore) as pre coding sequence of radiation-induced osteosarcoma DNA demonstrating a het viously described (38). After primary antibody immunoblotting, mem erozygous C —¿â€¢Ttransversion at the first base of codon 245, resulting in the substitution of serine for glycine. PCR primer sequences and direct sequencing branes were washed six times, 10 min each with 0.1% BSA in TBS. technique used are described in "Materials and Methods." Goat anti-mouse IgG conjugated to alkaline phosphatase (Promega) diluted 1:7500 in 0.1% BSA/TBS was added to Western immunoblots for 2 h at 24°C.Blots were again washed in 0.1 % BSA/TBS six times for 10 min each. Alkaline phosphatase dye indicator (Promega) was then added and incubated for 10-15 min. oc W co O « U Results

Tumor cells from seven radiation-induced osteo- and soft- 2-1 14Rb tissue sarcomas were examined for alterations at the Rb and p110! p53 loci. Experiments were performed on DNA extracted di rectly from the tumors and/or very early (third) passage cell lines. In the case ofp53, initial screening for mutations in exons 5-8 was done by PCR amplification (36) from genomic DNA, followed by SSCP analysis (35) of the amplified fragments. Fig. 2. Radiation-induced Rb Western immunoblots. Radiation-in Those samples with a variant pattern on SSCP analysis were duced tumor cells were lysed and protein was separated by sodium dodecyl sulfate analyzed by direct genomic sequencing. Abnormalities in p53 and 7.5% polyacrylamide. Transferred proteins underwent immunoblot with Rb were found in cell lines RIT-2, RIT-lb, and STSAR-5. In RIT- antibody PMB3-245 followed by alkaline phosphatase-conjugated anti-mouse 2, a heterozygous G-T was present in exon 7, IgG. Control cells include IMR-90 human fibroblasts and COS cells. Y79 (Amer ican Type Culture Collection), a retinoblastoma cell line without Rb protein, is which resulted in the substitution of serine for glycine. This used as a control. 6394

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1991 American Association for Cancer Research. SARCOMASTable RADIATION INDUCED HUMAN linesCell 1 Analysis of pii and Rb genes in radiation-induced human sarcoma cell mutations"Codon of second from lineRIT-lbRIT-2STSAR-5RIT-5RIT-6STSAR-28R1T-3HistologymalignancyPituitary primarytumorOsteosarcomaOsteosarcomaMalignant radiotherapy(years)76910201014p53Mutation2SO adenomaBreast deletion2458-base pair stopcodon,5' 8Glyend of exon cancerBreast C —¿TUnable -»SerCpRb*UnderphosphorylatedNormalNormalAbsentUnderphosphorylatedUnderphosphorylatedNormal fibrosis cancerHodgkin'sWilm's toamplifyNone HistiocytomaOsteosarcomaSpindle detectedNone sarcomaOsteosarcomaSynovialcell tumorDiffuse detectedND"*None celllymphomaRhabdomyosarcomaIntervallarge

sarcomaPrior detectedResultPremature " Exons 5-8 screened using PCR and SSCP; variants confirmed by direct genomic DNA sequencing. '' pRb, retinoblastoma protein. ' Total gene deletion on Southern analysis (data not shown). * ND, not done.

Underphosphorylated form of the retinoblastoma protein side of the region examined. Single substitutions (pllOKI>). Lanes 1, 5, and 6 show radiation-induced sarcomas within these regions have been shown to cause inactivation of RIT-2, RIT-3, and STSAR-5 with migration patterns of pi 12- wild-type p53 as growth suppressor (40). 114Khand pllORh that are similar to those patterns observed pi 10Kb was decreased in amount, pi 12-114Kb was absent for control cells COS, monkey kidney, and human fibroblast in three of seven tumors compared to control cells, and pRb line IMR-90. Lanes 2, 3, and 4 show an absence of pi 12Rhand was totally absent in a fourth tumor (Fig. 2 and Table 1). There pi 14Khand a diminished amount of pi 10Khin radiation-induced have been several mutant Rb proteins of M, ~\ 10,000 described sarcomas RIT-lb, RIT-6, and STSAR-28 compared to controls. in cells derived from bladder, prostate, and small-cell lung Lane 8 shows an absence of Rb protein in sarcoma RIT-5. Lane carcinomas (17, 22, 41). These mutant proteins appear to be 9 shows a negative control, retinoblastoma line Y79, which un- or Underphosphorylated forms of Rb with absent pll2Rb produces no Rb protein. Table 1 shows a summary of abnor and pi 14Rh.Recently, Mittnacht and Weinberg (42) and Tem- malities of tumor suppressor genes p53 and Rb in these radia pleton et al. (43) analyzed mutant Rb proteins and found that tion-induced sarcomas. Tumor cells derived from RIT-lb have they are associated with a decreased binding affinity for the abnormalities of both the p53 and Rb suppressor genes or gene nuclear compartment and were defective in their ability to products. All other tumor cell lines have an abnormality in become hyperphosphorylated. Also, the mutant pi lORb had an either p53 or Rb with the exception of cells derived from RIT-3. inability to induce in cultures transfected with an Rb gene that encoded the defective protein (45). Thus, the growth-suppressing ability of Rb protein may be altered in the Discussion radiation-induced sarcomas described hererin because of defects in status and a subsequent decrease in nuclear Given the apparent central role of p53 and Rb tumor sup pressor gene mutations in the genesis of "spontaneous" bone binding. and soft-tissue sarcomas as well as the high incidence of infield DNA alterations produced by ionizing radiation vary with the locus and system examined and do not have a consistent sarcomas after radiotherapy in patients with hereditary retino "fingerprint" (44). Therefore, although changes observed in the , we sought to examine radiation-induced human tu p53 DNA sequence and alterations in the Rb protein are con mors for alterations of the p53 and Rb tumor suppressor genes and/or their gene products. Six of seven tumors had abnormal sistent with findings after radiation exposure, these changes ities of one or both suppressor genes. may have occurred during tumor progression rather than as a In the case of the p53 gene, heterozygous mutations in exon direct result of radiation exposure. The fact that patients with 7 were observed in cell lines RIT-lb and RIT-2; both fall within hereditary retinoblastoma (who have a high rate of spontaneous previously described p53 mutational "hot spots" (7). Because sarcomas) develop with a shorter latency and early passage cell lines were used, it is possible that the apparent greater frequency within radiation fields supports the notion heterozygous mutations observed are actually homozygous that inactivation of the Rb gene may be accelerated by X-ray deletions with stromal contamination simulating a heterozy exposure (45, 46). Also, patients with the Li-Fraumeni syn gous state. Stromal contamination is unlikely, since karyotypic drome who inherit a mutated p53 alíelemay be at increased risk for radiation-induced tumors.4 analysis of these tumor lines failed to reveal evidence of normal cells (34). In addition we observed a total deletion of p53 in cell In summary, we demonstrate that sarcomas occurring after line STSAR-5 on Southern analysis. Thus, p53 mutations were ionizing radiation exposure contain abnormalities in tumor observed in three of six radiation-induced sarcomas; we did not suppressor genes p53 and Rb. Inactivation of these and/or other obtain DNA from STSAR-28 cells (Table 1). Our results may genes by X-rays may contribute to radiation carcinogenesis. underrepresent the true frequency of p53 mutations, since only exons 5 through 8 were examined. However, more than 90% of base substitution mutations in p53 discovered to date occur Acknowledgments in this region (39). Although the heterozygous nature of the We thank Drs. M. Simon and M. Post for generously providing mutations we describe herein suggests the possibility of a dom tissue specimens and Denise Adams for typing the manuscript. inant or a dominant-negative effect, our data do not exclude the possibility of additional mutations on the other alíeleout 4 D. W. Yandell, unpublished results. 6395

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David G. Brachman, Dennis E. Hallahan, Michael A. Beckett, et al.

Cancer Res 1991;51:6393-6396.

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