Proc. Nati. Acad. Sci. USAVol. 85, pp. 2106-2109, April 1988Biochemistry

Some retinoblastomas, osteosarcomas, and soft tissue sarcomas mayshare a common etiology

(recessive oncogenes/transcription/RB gene)

RALPH R. WEICHSELBAUM, MICHAEL BECKETT, AND ALAN DIAMONDUniversity of Chicago, Department of Radiation and Cellular Oqcology, Biological Sciences Division, 5841 South Maryland Avenue, Box 442,Chicago, IL 60637

Communicated by A. A. Moscona, December 7, 1987 (received for review September 17, 1987)

ABSTRACT DNA and RNA were extracted from primaryhuman osteosarcomas and soft tissue sarcomas obtained frompatients without retinoblastoma and were analyzed by hybrid-ization with a cDNA probe for RB mRNA; absence or alter-ations of the RB gene are associated with development of ret-inoblastoma. Most of the osteosarcomas or soft tissue sarcomasexamined by us did not express detectable levels of RB mRNA,whereas normal cells and epithelial tumor cells did. Oneosteosarcoma expressed a 2.4-kilobase transcript in addition toa normal 4.7-kilobase species. Our data suggest that transcrip-tional inactivation or post-transcriptional down-regulation ofthe RB gene may be important in the etiology of some osteosar-comas and soft tissue sarcomas as well as retinoblastomas.

Gene transfer experiments with the NIH 3T3 cell focusforming assay have demonstrated the presence of activatedtransforming genes in a variety ofhuman tumors. Oncogenesdetected in this way are thought to be dominant in theirmode of action (1-3). Another class of oncogenes has beendescribed in which tumor-predisposing mutations are reces-sive to wild-type alleles. These have been termed recessiveoncogenes, and the predisposing event may be loss-of-function mutations (4, 5). Human neoplasms such as retino-blastoma, Wilm tumor, uveal melanoma, rhabdomyosar-coma, and heptoblastoma have been hypothesized to resultfrom loss-of-function mutations (6-8). In Wilm tumor, arecessive mutation on chromosome 11 has been postulatedto be important in the etiology of this tumor (9). Dataindicate that the addition of a normal chromosome 11 toWilm tumor cells can result in the reversion of the tumorphenotype (10). Recessive oncogenes have also been de-scribed in Drosophila melanogaster where at least 24 suchgenes have been identified that cause tissue-specific tumors(11, 12).

It has been postulated that alterations of a gene at theretinoblastoma locus assigned to the q14 band of humanchromosome 13 (4, 13, 14) are required for tumor formationin retinoblastoma. Friend et al. (6) described the isolation ofa 4.7-kilobase (kb) cDNA that can be used as a probe todetect a chromosomal segment that, when altered or absent,predisposes to retinoblastoma. This cDNA, referred to asRB cDNA, hybridizes to a locus spanning at least 70 kb inthe q14 band of chromosome 13. The RB cDNA was used toprobe DNA from 40 retinoblastomas; of these, 30% showedabnormal restriction endonuclease cleavage patterns. Thiswork was extended by Lee et al. (15) whose analysis of theRB gene revealed that this gene contains at least 12 exonsdistributed over a region of 100 kb. Sequence analysis of thecloned cDNA revealed a single open reading frame thatpredicted a protein of 116 amino acids (15). Features of thepredicted amino acid sequence include a potential metal

binding domain similar to that found in nucleic acid-bindingproteins (15).

Individuals who inherit a mutant RB gene have a highincidence of spontaneous osteosarcomas as well as retino-blastoma, and it has been postulated that osteosarcomasoccur as a result of alterations resulting in the inactivation ofthe RB gene (16-19). Herein we describe results of experi-ments that extend this hypothesis to include several osteo-sarcomas and soft tissue sarcomas.

MATERIALS AND METHODS

Human tumor specimens were obtained from patients under-going surgical procedures; the clinical data are summarizedin Table 1.

Southern Analysis. DNA was extracted as described (20)after the tissue samples were homogenized in a 7.0-mlDounce homogenizer. DNA (10 ,ug) was digested with Hin-dIII restriction enzyme and was electrophoresed through1.0% agarose gels and transferred to nitrocellulose with 20 xSSC (3.0 M NaCl/0.3 M sodium citrate, pH 7.0). Hybridiza-tion with 32P-labeled 3.8-kb RB cDNA (prepared by therandom primer procedure) (21) was performed for 16 hr at370C in a solution containing 0.8 M NaCl, 0.1 M Mops,denatured salmon sperm DNA at 100 pug/ml, 0.1% sarcosine,and 5x Denhardt's solution (1x Denhardt's solution =0.02% polyvinylpyrrolidone/0.02% Ficoll/0.02% bovine se-rum albumin). Filters were washed twice at 650C in 0.2xSSC/0.1% sarcosine/0.05% sodium pyrophosphate and ex-posed to Kodak XAR film with an intensifying screen at- 850C.RNA Gel Blot Analysis. Total cellular RNA was isolated as

described (22). Ten-microgram samples of each RNA wereelectrophoresed on 1% agarose gel containing 2.2 M form-aldehyde (20) and were transferred to GeneScreenPlus (NewEngland Nuclear) by the recommended procedure of themanufacturer. Random primer probes of the 3.8-kb EcoRIRB cDNA fragment were synthesized as described (21).Hybridization was performed for 16 hr at 420C in 50%(vol/vol) formamide, 10% (vol/vol) dextran sulfate, 1%NaDodSO4, and 1 M NaCl. The blots were washed twice for5 min at 250C in 2 x SSC, twice for 30 min at 600C in 2 xSSC, and twice for 30 min in 0.1 x SSC at room temperature.The membranes were exposed to Kodak XAR film betweentwo intensifying screens at - 850C.

RESULTS

DNA and RNA were obtained from primary human osteo-sarcomas and soft tissue sarcomas. A description of thesetumors is presented in Table 1. Included in these experi-ments as controls are RNA from the normal fibroblast cellstrain 1522 and DNA from the squamous cell carcinoma cellline SCC-12 B.2 (23).

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Proc. Natl. Acad. Sci. USA 85 (1988) 2107

Table 1. Summary of primary tumor samplesPatient

Age, PrimarySample Sex years Disease disease site

21 Female 74 Soft tissue sarcoma* Left foot22 Male 17 Osteosarcoma Left femur23 Female 52 Osteosarcoma Left femur36 Female 23 Osteosarcomat Left tibia116 Female 19 Osteosarcoma Left femur118A Male 46 Soft tissue sarcoma* Stomach32 Female 51 Soft tissue sarcomat Right thigh*Leiomyosarcoma.tThis sample was a lung metastasis.tMalignant fibrous histiocytoma.

Fig. 1 shows the pattern of hybridization of the DNA fromthe primary osteosarcoma 116, a cell line derived from theosteosarcoma 22, two primary soft tissue sarcomas (sar-comas 21 and 32), and SCC-12 B.2 cells probed with the3.8-kb RB cDNA representing the 3' end of the RB gene (6,15). Although some bands on the lanes containing DNA fromthe osteosarcomas and soft tissue sarcomas were reduced inintensity compared to the SCC-12 B.2 control DNA, the2.3-, 4.2-, 5.3-, 7.1-, 7.7-, 8.3-, and 9.1-kb bands were visiblein all samples on longer exposure of the filters. The relativereduced intensity of these bands may reflect deletions orrearrangements of the RB gene in DNA from some tumors,possibly in conjunction with hemizygosity. However, DNA

- 9.1- 8.3- 1.7- 7.1

-5.3

-4.2

.At

1 2 3 4 5

FIG. 1. Southern blot analysis showing restriction enzyme

cleavage patterns of DNA from an epithelial tumor cell line, threeprimary sarcomas, and a cell line derived from an osteosarcoma.DNA was isolated from osteosarcoma cell line 22 (lane 1), primary

soft tissue sarcomas 21 (lane 2) and 32 (lane 3), primary osteosar-coma 116 (lane 4), and a squamous cell carcinoma cell line SCC-12B.2 (lane 5). DNAs were digested with HindIll, electrophoresed in1% agarose gels, transferred to GeneScreenPlus filters, and hybrid-ized to the 3.8-kb RB probe. Molecular weights (x 10-O) of theresulting bands were determined by comparison to the mobility ofHindIl-digested A DNA, which was used as molecular weight stan-dards. DNA was not obtained from tumors 23, 36, or 118A.

degradation cannot be ruled out as an alternative explana-tion, especially in view of the observation that DNA from 9of 10 osteosarcoma cell lines exhibited a normal HindIIIrestriction enzyme pattern when probed with the 3.8-kb RBcDNA (T. Dryja, personal communication).

Total RNAs prepared from osteosarcomas, soft tissuesarcomas, and control cells were analyzed on RNA gel blotsby using the 3.8-kb EcoRI fragment from the RB cDNA (6,15)as the hybridization probe (Fig. 2A). Since the yield of RNAfrom tumor samples was generally low, poly(A)+ RNA iso-lation was not attempted. Total RNA from the fibroblast cellstrain 1522 (Fig. 2A, lanes 1 and 6) contains a normal 4.7-kbmRNA species similar to what has been demonstrated formpst tissues and tumors (6, 15). However, the 4.7-kb RBtranscript was not detected in the RNA of osteosarcomasamples 23 and 116 (Fig. 2A, lanes 2 and 4), although the sameamount of RNA was placed in each lane and although actinmRNA was detected on the same filters by hybridization to anactin probe (Fig. 2B). RNA from primary osteosarcoma 23also failed to demonstrate detectable levels of hybridization tothe 3.8-kb RB probe (Fig. 2A, lane 2); however, reducedhybridization of this sample to the actin probe suggested thatsome degradation had occurred (Fig. 2B, lane 2). Osteosar-coma 36 (Fig. 2A, lane 3), which expressed a 4.7-kb RNA,also expressed an abnormal species migrating at -2.4 kb. It isinteresting that 4.0-kb RB transcripts were observed in threeof six retinoblastomas examined by Lee et al. (15), suggestingthat truncated transcripts may accompany or contribute totumor development.RNA isolated from soft tissue sarcomas 21 and 118A (Fig.

2A, lanes 7 and 5) also failed to hybridize to the RB cDNAprobe. It is unlikely that the absence of hybridization to theRB cDNA fragment was due to degradation of tumor RNA.This was determined by the similarity of the ethidiumbromide staining patterns on RNA agarose gels and by thehybridization of the same filters to an actin probe (Fig. 2B).Thus, lack of detectable transcripts from the RB gene is acommon feature of osteosarcoma and soft tissue sarcomas,as well as retinoblastomas.

DISCUSSIONWe have analyzed DNA and RNA in spontaneous osteosar-comas and soft tissue sarcomas obtained from patientswithout retinoblastoma. It is noteworthy that this analysiswas performed on nucleic acid derived directly from thetumors. Therefore, these observations do not involve arti-facts that complicate studies of tissue cultures or of estab-lished cell lines that may not be representative of theirtumors of origin.The data in the Southern blot in Fig. 1 indicate that the RB

gene in, at least, somne tumors may have undergone structuralalterations, as evidenced by bands of reduced relative inten-sity in some lanes. Longer exposure of this blot demonstratesthe presence of all the bands observed in control DNA. Thepresence of these bands may be due to contaminating DNAfrom normal cells included in the tumor sample or simplyreflect DNA degradation. The latter explanation seems lesslikely as the loss of intensity appears to occur specifically incertain bands. RNA from all tumor samples, however, fail tocontain detectable levels of RB transcripts. While deletionsand gross rearrangements are obvious candidates for alter-ations that reduce or eliminate RB transcription, it is possiblethat point mutations or small deletions and insertions mayaffect transcription or post-transcriptional regulation. Meth-ylation of the RB gene might also reduce transcription (24,25), and it is also conceivable that the genetic defects in thetumors studied occur in an as yet unidentified gene that af-fects the transcription of the RB gene.

Biochemistry: Weichselbaum et al.

'Moiwaw

i...W4111,

'10 4'W, - 2.3

2108 Biochemistry: Weichselbaum et al.

A

4.7-

2.4-

-28s

-18s4.7-

1 2 3 4 5

- 28s

-18s

6 7

B

-28s-18s -28s

-18s

2 3 4 5

6 7

FIG. 2. RNA gel blot analysis indicating absence of detectable RB transcripts in RNA from primary osteosarcomas and soft tissuesarcomas. (A) Total RNA (10 Ag per lane) was loaded from the normal fibroblast cell strain 1522 (lanes 1 and 6), from primary osteosarcomas23 (lane 2), 36 (lane 3), and 116 (lane 4), and from primary soft tissue sarcomas 118A (lane 5) and 21 (lane 7). A band corresponding to the 4.7-kbRB mRNA is detectable in the RNA sample from the fibroblast cell strain 1522, whereas the RNA sample from osteosarcoma 36 expresses the4.7-kb species as well as an abnormal species migrating at -2.4 kb (indicated by arrows). The data presented in this figure were obtained fromtwo gels, lanes 1-5 representing one gel and lanes 6 and 7 another. (B) The same filters shown in A were hybridized to a mouse actin probeindicating that, with the exception of primary osteosarcoma 23, all lanes were loaded with approximately the same amount and quality of RNA.The mobilities of the 18S and 28S rRNA species are indicated. RNA was not obtained from tumors 22 or 32.

Hereditary retinoblastoma patients who are cured of theirtumors are at high risk of developing secondary neoplasms,particularly osteosarcoma. These tumors occur both withinand outside of the field of radiation therapy (16-19). Devel-opment of osteosarcoma in patients carrying an altered RBallele suggested that this gene plays a role in the etiology ofboth tumors. Dryja et al. (16) used restriction fragmentlength polymorphism analysis of chromosome 13 to study 15osteosarcoma cell lines derived from tumors arising in pa-tients without retinoblastoma. DNAs from three of theselines were homozygous at every chromosome 13 locus andpresumably homozygous for any recessive tumor predispos-ing allele. Hapsen et al. (17) also employed restrictionfragment length polymorphisms to examine seven osteosar-comas. Three patients had osteosarcoma associated withretinoblastoma, two of which were associated with loss ofheterozygosity of chromosome 13. Four patients had spo-radically occurring retinoblastoma of which three were as-sociated with loss of heterozygosity at chromosome 13. In allinstances the loss of heterozygosity included the region of13q14 (17). Friend et al. (6) analyzed DNA from 8 osteosar-comas and found that some had deletions or rearrangements.Expression of the RB gene was analyzed in only one case ofosteosarcoma, and it was not detectable when compared tocontrol embryonic retina (6).

A normal 4.7-kb RB transcript has been shown in a varietyof rat tissues including adult liver, brain, kidney, ovary, andspleen as well as fetal liver, brain, and kidney (15). Humansamples shown to express the 4.7-kb transcript include retinalcells, small cell carcinoma of the lung, neuroblastoma, me-dulloblastoma, renal carcinoma, and melanoma (6). Our datasuggest that lack of expression of the RB gene in osteosar-coma and soft tissue sarcoma may be common, since we didnot detect evidence of RB gene transcripts in most of thetumors examined. This conclusion, if further confirmed, mayhave important implications concerning the etiology of thesetypes of neoplasms. Another possibility to consider is thatalterations or lack ofexpression oftheRB gene occur after thetumor develops and are associated with the particular tumorswithout being a cause of the malignant transformation. It isintriguing that radiation-induced, as well as spontaneous,osteosarcomas are common in patients with hereditary reti-noblastoma (18, 19). Radiation is known to induce DNAdeletions (26); thus, an important step in radiation-inducedcarcinogenesis in these patients may be deletion or inactiva-tion of a recessive oncogene. This may be an important factorin radiation-induced carcinogenesis in general.

We thank R. Weinberg and T. Dryja for the RB cDNA clone, A.Moscona for critical reading of the manuscript, T. Dryja and J.

Proc. Natl. Acad. Sci. USA 85 (1988)

Proc. Natl. Acad. Sci. USA 85 (1988) 2109

Rowley for helpful discussions, and M. Simon for tumor biopsysamples. This work was supported by grants from the NationalInstitutes of Health to R.R.W. and by the Passis Fund.

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