(CANCER RESEARCH 48, 5251-5255, September 15. 1988)

Activated H-ras Oncogenes in Human Kidney Tumors1

Jun Fujita, Matthias H. Kraus, Hitoshi Onoue, Shiv K. Srivastava, Yoshitaka Ebi, Yukihiko Kitamura, and Johng S.Rhim2

Institute for Cancer Research, Osaka University Medical School, Nakanoshima, Kita-ku, Osaka 530, Japan [J. F., H. O., Y. E., Y. K.], and Laboratory of Cellular andMolecular Biology, National Cancer Institute, N1H, Bethesda, Maryland 20892 fM. H. K., S. K. S., J. S. R.J

ABSTRACT

Two H-ras oncogenes were detected by NIH/3T3 transfection assayout of 16 primary kidney tumors, 15 renal cell carcinomas (RCC), andone transitional cell carcinoma in 16 patients. Analysis of ras M, 21,000protein suggested single point mutations within codon 12 and 61 in eachcase. The restriction endonuclease analysis of H-ras gene at codon 12confirmed this in one of them, and the remaining 15 tumors did not havea mutation at this site. DNAs from the noncancerous portions of thekidney with codon 12 mutated tumor, but not leukocytes from the samepatient, showed an abnormal resistance to the endonucleases Mspl andHpall, suggesting a presence of codon 12 mutated H-ras gene in thenoncancerous cells. No amplification of ras genes was detected in the 16tumors analyzed. In one of eight tumors from patients heterozygous forH-ras related Ram\\\ restriction fragments, one alíelewas lost in thetumor but not in the noncancerous portion of the same kidney. Althoughcytogenetic studies have previously suggested nonrandom involvement ofc-raf-l gene in RCC, no abnormality in the size nor amount of ruftranscript was detected in the 15 RCCs. Our results thus indicated thatthe genetic lesions affecting ras genes do occur in human RCC, andprobably serve as one of multisteps in the carcinogenic process.

INTRODUCTION

The ras oncogenes have been associated with many types ofanimal and human tumors and were assumed to play a role inthe development, maintenance, or progression of malignancies(1, 2). The ras genes acquire the transforming capacity eitherby single point mutations that cause an alteration at amino acidpositions 12, 13, or 16 of the M, 21,000 ras gene product, orby the overexpression of the normal ras p213 (3, 4). It has beenestimated that 5-20% of all human malignancies contain mutated ras genes (1, 3). Previously, we analyzed human urinarytract tumors for such mutationally activated ras oncogenes bycombined use of NIH/3T3 transfection assay and the restrictionendonuclease analysis at codons 12 and 61 of H-ras gene, andindicated that H-ras oncogenes were activated in 10% of thetumors (5). Although we demonstrated that these point mutations were acquired somatically in the tumor cells (6), it remainsto be determined at what stage of carcinogenic process rasoncogenes are activated and to what extent they affect theclinical course of the patient.

RCC is the most common malignant disease arising from thekidney. It is potentially curable when detected early, but in mostcases it is detected late and responds poorly to treatment. Somepercentage of RCC are familial and are suspected to be heritable(7). Although rearrangements or loss of alíelesof loci involvingthe short arm of chromosome 3 (3p) have been identifiedfrequently in the tumors of both familial and sporadic cases (7-10), most familial RCC patients show no constitutional chromosomal abnormality (7, 11), suggesting that some other her-

Rcceived 1/21/88; revised 5/13/88; accepted 6/17/88.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

' Supported in part by Grants-in-Aid from the Ministry of Education, Scienceand Culture and the Ministry of Health and Welfare of Japan.

2To whom requests for reprints should be addressed.'The abbreviations used are: p21, M, 21,000 protein; RCC, renal cell carci

noma; SDS, sodium dodecyl sulfate.

itable predisposing factors may be involved with the rearrangement or the loss of alíelesat the 3p acquired duringtumor development. Since the involvement of mutated rasoncogenes has not been assessed in familial or sporadic casesof RCC, and human raf (c-raf-l) gene, which has been mapped

on chromosome 3p, may be affected by the rearrangement ( 12),we analyzed 16 cases of primary kidney tumors, including ISRCCs, for the presence of mutationally activated ras oncogenes,loss of one H-ras alíele,and an abnormal expression of raf

gene.

MATERIALS AND METHODS

Patientsand Tumors. A total of 16 primary kidney tumors (RMIT—RM8T and RS1T-RS8T), 10 on the left and 6 on the right, were

surgically obtained from 16 patients, 10 males and 6 females. The agesof the patients were 61.1 ±10.2 (SD). Immediately after removal ofthe whole kidney with tumor, the tumor and grossly normal portion ofthe kidney were dissected, frozen in liquid nitrogen, and stored at—80°Cuntil extraction of DNA. The tumors were all RCCs except one

transitional cell carcinoma (RM4T), and 10 of them had invasions intothe surrounding tissues or metastasis.

Preparation of DNA and RNA. High molecular weight DNAs wereextracted from pulverized tissues with lysis buffer containing 1% SDS,0.1 M Tris hydrochloride (pH 7.5), 50 mM EDTA, and 200 ¿ig/mlproteinase K. DNA was purified by repeated extractions with phenol/chloroform and precipitation in 70% ethanol after RNase treatment asdescribed (6).

Total cellular RNAs were extracted from tissues by the guanidinethiocyanate/cesium chloride method, and polyadenylated RNA wasobtained by passing the total RNA through an oligodeoxythymidylate-

cellulose column (13).DNA Transfection. NIH/3T3 cells were grown in Dulbecco's modi

fied Eagle's medium supplemented with 10% calf serum, and were used

as recipients for DNA transfection. DNAs were transfected by thecalcium phosphate coprecipitation technique as previously described(6). The numbers of transformed foci were counted 21-28 days after

transfection.DNA and RNA Blotting Analyses. DNA samples (20-50 ¿<g)were

digested with appropriate restriction endonucleases under the conditions supplied by the manufacturer, electrophoresed on 1.0 or 1.8%agarose gels, and transferred to nitrocellulose filters. They were hybridized to nick-translated 32P-labeled human DNA probes specific to each

member of raígene family or raf gene (pHEl, kindly provided by Dr.G. Mark) under stringent conditions as described (5, 14).

RNA extraction and blotting were done as described by Thomas (15)with slight modifications. Samples of 5 ng of polyadenylated RNA wereelectrophoresed on 1.0% formaldehyde agarose gels (14), blotted ontonitrocellulose filters, and hybridized to human raf and .¡actin (16)probes under the same conditions as those for DNA blot hybridizationwith the use of 50% formamide.

Immunoprecipitation of ras p21. Metabolic labeling of cells with[35S]methionine and immunoprecipitation of raíp21 by using a mono

clonal anti-p21 antibody (Y13-259; Ref. 17) was done as described (18).Labeled cell extracts, 5 x 107cpm per sample, were immunoprecipitatedand analyzed on 12.5% SDS/polyacrylamide slab gels. Y13-259 anti

body is specific for the p21 proteins, mutated or normal, encoded by 3members of the human raígene family.

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ACTIVATED H-ras ONCOGENES IN HUMAN KIDNEY TUMORS

RESULTS

Detection of H-ras Oncogenes by DNA Transfection Assay.When 16 kidney tumors were analyzed by DNA transfectionassay by using NIH/3T3 cells as recipients, two (RM4T andRS7T) scored as positive, giving transformed foci at an efficiency of 0.01 and 0.03 foci/^g of DNA, respectively. In eachcase, the transforming activity was observed in two independentexperiments, survived a second cycle of transfection, and humanrepetitive sequences were demonstrated in all first- and second-cycle transfectants analyzed (data not shown), indicating thepresence of oncogenes in the original tumors. DNAs from theremaining 14 tumors, however, did not give any transformedfoci after transfection of 640 ng of DNA/sample. RM4T wastransitional cell carcinoma of the left kidney with lymph nodemetastasis found in a 76-year-old male patient. RS7T was RCCof the left kidney found in a 52-year-old female patient. Neitherof them had any previous histories of malignancy nor of exposure to mutagens, and their family histories suggested no specialtendency to develop malignancies.

Since ras oncogenes have been most frequently detected byNIH/3T3 transfection assay (1, 3), DNAs from RM4T- andRS7T-transformed cells were hybridized with nick-translatedprobes specific to each member of the ras gene family. Asshown in Fig. 1/1, only the H-ras-specific probe detected DNAbands other than the mouse endogenous bands in both transformants (Lanes I and 3), indicating that the oncogenes inRM4T and RS7T were activated alíelesof the H-ras gene.

Altered Electrophoretic Mobilities of ras p21. Altered electro-phoretic mobilities of p21 on SDS/polyacrylamide gels couldbe classified as slow- or fast-moving species in comparison tonormal human or mouse p21 bands, the former suggesting apoint mutation within codon 12 and the latter within codon 61of the ras gene (18). RM4T transformants and RS7T transform-ants were analyzed for p21 with such altered electrophoreticmobilities by using an anti-p21 antibody Y13-259 (17). Asshown in Fig. IB, the former (Lane 4) yielded p21 that migratedfaster than normal p21 bands (Lane 5), which usually consistedof two bands, and the latter (Lane 6) yielded p21 that migratedslower than normal p21 bands. RM4T and RS7T were likelyto have H-ras oncogenes activated by point mutations withincodon 61 and codon 12, respectively.

Restriction Endonuclease Analysis of H-ras Gene at Codon12. Since an alteration at codon 12 of human H-ras gene leadsto loss of a restriction site for Hpall/Mspl digestion (6, 19),and analysis of p21 suggested such a mutation in RS7T tumor,

1 2 3 456

r—Ar P21

Fig. 1. Identification of H-ras oncogenes in human kidney tumors and alteredelectrophoretic mobilities of ras p21. A, high molecular weight DNAs weredigested with BamHl and hybridized to a H-ras-specific pKYl probe (5). DNAswere from NIH/3T3 cells transformed by the kidney tumor RM4T (Lane 1) orRS7T (Lane .?) DNA, and NIH/3T3 cells (Lane 2). Coelectrophoresed HindlU-digested A cl857 DNA served as molecular weight standards and are indicatedby arrowheads, B, NIH/3T3 cells transformed by the kidney tumor RM4T (Lane5) were labeled with ("S]methionine. After immunoprecipitation with an anti-p21 antibody (Y13-259), electrophoretic mobilities of p21 were analyzed asdescribed (18). Bands of p21 with normal electrophoretic mobility are shown by

16 primary tumor DNAs were analyzed by this moleculardiagnostic method. As expected, all tumor samples exceptRS7T demonstrated a normal 355 base pair band only (Fig. 2,Lane 2 for RM4T tumor; other data not shown) like humankidney DNA (Lane 1). RS7T tumor (Lane 5) and its transfec-tant (Lane 6) yielded the expected 411 base pair band like theT24 transfectant (Lane 7), confirming the presence of a pointmutation within codon 12 of H-ras gene in RS7T tumor. Ratherunexpectedly, normal portion of the resected kidney harboringRS7T tumor (RS7N; Lane 4) also gave 411 base pair band inaddition to 355 base pair band, while leukocyte DNA from thispatient (Lane 3) showed a normal 355 base pair band only.

Mutated H-ras Gene in Noncancerous Kidney Cells. AlthoughRS7T tumor was encapsulated, and the normal portion resected(RS7N) was more than 5 cm distant from the tumor, there stillremains a possibility of unsuspected spread of the tumor intoRS7N. Therefore, slices were cut across the middle and bothedges of a small piece (3x3x4 mm) from the frozen RS7Ntissue and examined histologically, while the remaining portions of the piece (RS7NA and RS7NB) were used for DNAextraction. In order to assure the completeness of restriction, alarge excess of Mspl and Hpall endonucleases (60 units each/tig DNA) were used, and some samples were mixed with clonedX-phage DNA before digestion. As shown in Fig. 3, no contaminating tumor cells were observed in the normal portions of thekidney analyzed. However, as Fig. 4B shows, both RS7NA(Lanes 2 and 5) and RS7NB (Lanes 3 and 6) showed abnormal411 base pair bands like RS7T tumor (Lanes 4 and 7) and itstransformant (Lane 8), while normal kidney DNA (Lane 1)showed only 355 base pair bands. The identical restrictionpatterns of X-phage DNAs in Lanes 1 to 4 of Fig. 4A indicatedthat these samples were equally well digested. Furthermore,Fig. 4A confirmed the absence of contamination by X-phages inRS7N as well as RS7T DNA samples.

Loss of an 11-ra.yAlíele.Although we have previously foundan amplified K-ras gene in a bladder carcinoma (5), no amplification or rearrangements involving any of the ras genes wasdetected in the 16 kidney tumors (data not shown). However,an abnormality in H-ras gene was found in one of them. H-rasgene (c-H-ras-1) shows polymorphism by BamHl restrictionendonuclease analysis due to the variations in the number ofreiterations of a small repeated sequence located 3' to its codingregion (20). Thus the DNA from individuals who are homozy-gous for this locus contains a single H-ras fragment, while DNAfrom heterozygous individuals contains two H-ras fragments asshown in Fig. 5. In the present study, 8 of 16 patients wereheterozygous for the H-ras gene locus. When the intensity ofthe two fragments representing two alíeleswas compared toeach other, one fragment was repeatedly found to react with

1 2345678

*"^ A.

—4T••-411-•-355

Fig. 2. Restriction endonuclease analysis of human 11 ras gene at codon 12.High molecular weight DNAs were digested with Mspl and Hpall, and hybridizedto an H-roi-specific pKYI probe (5). DNAs were from noncancerous (RM4N)and cancerous (RM4T) portions of the kidney from a patient (RM4) (Lanes 1and 2, respectively); leukocytes, noncancerous (RS7N) and cancerous (RS7T)portions of the kidney from a patient (RS7) (Lanes 3, 4, and 5, respectively);NIH/3T3 cells transformed by RS7TDNA (Lane 6) or T24 oncogenes (Lane 7);and NIH/3T3 cells (Lane 8). Note the presence of 411 base pair fragment inLanes 4 and 5.

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ACTIVATED H-««ONCOGENES IN HUMAN KIDNEY TUMORS

IFig. 3. Absence of metastasis in the non-

cancerous portion of the kidney used for DNAextraction. Formalin-fixed, paraffin-embeddedtissue sections were made from frozen tissuesand stained with hematoxylin and eosin. Left,RS7T kidney tumor showing clear cell carcinoma with scarce stromal cells (x 100); middle, RS7NA kidney tissue obtained from thesame kidney as RS7T tumor. Note the absenceof metastatic cancer cells (x 100); right, lowmagnification of RS7NA tissue (x 40).

1 23456789

355-»'

111-355^1 ..

some 3p, and shift of the c-raf-l locus in some sporadic casesof RCC (12). Since rearrangements can affect expressions orthe biochemical functions of protooncogene (21 ), we analyzedthe size and amount of raf transcript in the 16 kidney tumors.The mRNA was easily detected in all the tumors (Lanes 3-18)and normal kidneys (Lanes 1 and 2) as well as human placenta(Lane 19), and there was no obvious abnormality in the size oramount expressed in these samples. In concert with this, noamplification of ra/gene was detected in the tumor DNAs (datanot shown).

Fig. 4. Resistance to restriction by Msp\/Hpa\\ endonucleases at codon 12 ofthe H-ras gene in noncancerous kidney cells. High molecular weight DNAs weredigested with a large excess of Mspl and ///mil endonucleases, and successivelyhybridized to a X-phage DNA (A) and a H-raj specific pKYl probe (B). DNAswere from cloned X-phage and normal kidney RSI N (Lane /); X-phage andnormal kidney RS7NA or RS7NB (Lanes 2 and 3, respectively); X-phage andkidney tumor RS7T (Lane 4); RS7NA (Lane 5); RS7NB (lane 6); RS7T (Lane7); NIH/3T3 cells transformed by RS7T DNA (Lane 8); and NIH/3T3 cells(Lane 9). Coelectrophoresed DNA fragments of//aelll-digested 0X174RF DNAserved as molecular weight standards and are indicated by arrowheads.

12 34 56 7 8

-23.1

«9.4

«6.6

< 4.4

Fig. 5. Restriction fragment length polymorphism and loss of one H-raj-1alíelein human kidney tumors. Paired samples of DNA from noncancerous andcancerous portions of the same kidney were digested by BamHl, and analyzed bySouthern blot technique. DNAs were from kidney tumors RM4T (Lane 2), RS1T(Lane 4), RS5T (Lane 6), and RS6T (Lane 8). Lanes I, 3, 5. and 7 were DNAsfrom normal portions of the kidney harboring tumors RM4T, RS1T, RS5T. andRS6T, respectively. Coelectrophoresed DNA fragments of //indlll-digested Xc 1857 DNA served as molecular weight standards (shown in kilobase pairs).

the probe weaker than the other (RM6T; Lane 8). Both fragments were equally intense in the noncancerous portion of thekidney from the same patient (RM6N; Lane 7). RM6T tumorwas a RCC invading an outside renal capsule found in the rightkidney of a 67-year-old female patient.

Expression of raf Gene. Recently a cytogenetic study demonstrated the presence of a rearrangement involving chromo-

DISCUSSION

There are about 40 protooncogenes and oncogenes, and byone means or another, damage to diverse protooncogenes hasbeen indicated in the genesis of human tumors (21). We demonstrated here that 2 (RM4T and RS7T) of 16 kidney tumors,including IS RCCs and one transitional cell carcinoma(RM4T), contained H-ras oncogenes capable of transformingNIH/3T3 cells in vitro. Analysis of electrophoretic mobilitiesof raj p21 (18) suggested that the activating mutations residedwithin codon 61 (RM4T) or codon 12 (RM7T) of respectiveH-ras genes, which was consistent with the result of the restriction endonuclease analysis of 11 MS gene at codon 12. Thesefindings strengthened the notion that ras oncogenes are themost frequently activated oncogenes in human tumors, andcodons 61 and 12 are the two major hot spots for their muta-tional activations, the monotony of the mutations probablyrepresenting a consequence of biological selection. They furthersuggest the potential usefulness of p21 analysis in screening alarge number of human materials for activated ras oncogenes.The frequency of point-mutated ras oncogene in the kidneytumors was thus almost the same as those reported for otherhuman malignancies, and much less than those found in somechemically induced animals tumors (3, 5, 22, 23). Whether thisdifference in frequency is attributable to strict protocols andstrong carcinogens used in the animal studies and the diverse,mostly unknown etiological agents encountered under variousconditions in human situations, remains to be determined.

Activated ras oncogenes so far have proven to be somatic inorigin, restricted to tumor tissues, by DNA transfection assayor restriction endonuclease analysis (3, 5). The restriction endonuclease analysis of H-ras gene at codon 12 is based on thefact that both restriction endonucleases Mspl and Hpall rec-

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ognize the sequence C-C-G-G encompassing codon 12 of thehuman H-ras protooncogene (19), and use of both enzymesreduces the effect of methylation at this site (14). Thus anyalterations in the codon 12 of H-ras gene (normally G-G-C)affecting its encoded amino acid would lead to loss of the Msp\/Hpa\\ site (5). Consistent with the somatic origin, the pointmutation found in RS7T tumor was not detectable by this assayin the leukocyte DNA from the same patient. However, theDNA sample purified from noncancerous portion of the kidneybearing RS7T tumor repeatedly showed resistance to Mspl/Hap\\ digestion at H-ras codon 12 like the RS7T tumor DNA.In order to rule out microscopic metastasis, we repeatedlyextracted DNAs from very small pieces of noncancerous kidneytissue (RS7N) and confirmed the absence of tumor cells histo-logically. Furthermore, the completeness of endonuclease digestion was assured by simultaneous complete digestion of X-phageDNA added to the RS7N DNA samples. Therefore, the presentresults strongly suggest, although not confirmed by molecularcloning and sequencing of the gene, that the histologicallynormal kidney cells as well as tumor cells from a patient withRCC contained a codon 12-mutated H-ras gene which wasapparently not transmitted in a germ line. This may not bewithout precedents. Sakamoto et al. (24) did DNA transfectionassay by using NIH/3T3 cells as recipients and found a noveltransforming gene hst in the noncancerous stomach mucosa aswell as in the stomach cancer of the same patient. Furthermore,results from in vivo animal studies suggested that activation ofras genes can play a role in a mult ¡stepprocess of carcinogen-esis, perhaps serving as an initiating event, but does not sufficefor tumorigenesis (22, 23, 25). Since the normal 355 base pairH-ras fragment was always present in MspI/A/pall-digestedRS7T tumor DNA and only a few contaminating stromal orblood cells were found in the tumor tissue under microscope,the majority of RS7T tumor cells were probably heterozygousat the H-ras gene, one mutated and the other normal. Furtherinvestigations are necessary to determine the nature and thenumber of additional genetic changes involved in the conversionof a noncancerous kidney cell with probably point-mutated H-ras gene into a malignant cell.

Damages to chromosomes, translocations, deletions, and abnormal amplifications, can affect either the expression or thebiological function of protooncogenes, and have been especiallyrevealing clues to oncogenes (21). Recently several studies haveshown that normal sequences on the short arm of chromosome11 (lip) are lost during the development of some tumors,including Wilms' kidney tumors and bladder tumors (21, 26,

27), and somatic loss of heterozygosity for this sequence, whichmight be involved in control of tumorigenic expression, ishypothesized to result in homozygosity for a recessive mutantalíele.To detect such a somatic loss of chromosome lip sequences in the tumor, we exploited DNA polymorphism onchromosome 1Ip by using a cloned H-ras gene as a probe (26),and compared DNAs from malignant and nonmalignant kidneycells from the same patient. In one of the eight tumors derivedfrom patients heterozygous at this locus, loss of one H-ras alíelewas found. This finding suggested that recessive genetic changesinvolving 1Ip might contribute to the development of humankidney tumors, although they were not absolutely required forprogression to malignancies.

In both the familial and sporadic cases of RCC, nonrandominvolvement of chromosome 3p, on which the c-raf-\ protooncogene is mapped, has been suggested (7-11). By using short-

term in vitro cultures, Teyssier et al. ( 12) recently demonstrateda rearrangement of the 3p in 4 of 6 human RCCs, and in 2 of

them the shift of c-raf-i gene to the breakpoint region was alsoidentified. Furthermore, a rearranged ra/gene has been foundas an oncogene by NIH/3T3 transfection assay (28). Sincerearrangements involving protooncogenes could result in theirenhanced expression as exemplified by myc gene in Burkittlymphoma, or structural alterations in the encoded protein asexemplified by fusion of abl and ber genes in Ph' chromosome

(21), we surveyed 15 RCCs for the presence of enhanced expression or abnormal size of ra/mRNA. However, no abnormalityin ra/gene expression was detected. A transforming raf oncogene was not detected in them by NIH/3T3 transfection assay,either, suggesting that alterations in c-raf-\ gene does not playa major role in the pathogenesis of human RCC. Elevatedexpression of c-myc and an apparent activation of c-raf-\ genehave recently shown in radioresistant, noncancerous skin fibro-blasts from a cancer-prone family (29).

The present study demonstrated that activation of ras oncogenes and a loss of one c-H-ras-1 alíeleon chromosome lipdid occur, although at a low frequency, in the primary kidneytumors. A combination of genetic lesions involving severaloncogenes probably contribute to tumorigenesis in the kidney,and the presence of mutated H-ras gene in the noncancerouscells as suggested here is consistent with this notion. However,most genetic lesions remain unidentified in the majority ofhuman tumors, and even some identified lesions could representeffect rather than the cause. Further studies are mandatory toidentify the genetic lesions having major intrinsic effects on thedevelopment of kidney tumors and to clarify the molecularmechanisms of their actions.

ACKNOWLEDGMENTS

The authors acknowledge Dr. Michel Lieber, Mayo Clinic, for providing tumor tissue. Dr. G. Mark for providing human c-raf complementary DNA probe pHEl, and Dr. S. A. Aaronson, National CancerInstitute, for helpful discussions.

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1988;48:5251-5255. Cancer Res   Jun Fujita, Matthias H. Kraus, Hitoshi Onoue, et al.  

Oncogenes in Human Kidney TumorsrasActivated H-

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