(CANCER RESEARCH 50. 6139-6145. October 1. 1990]

K-ras Activation in Neoplasms of the Human Female Reproductive Tract

Takayuki Enomoto, Masaki Inone, Alan O. Perantoni, Naoki Terakawa, Osanni Tanizawa, and Jerry M. Rice1

Laboratory of Comparative Carcinogenesis, National Cancer Institute, Frederick Cancer Research and Development Center, Frederick, Maryland 21702-1201 [T. E.,A. O. P., J. M. R.], and Department of Obstetrics and Gynecology, Osaka University Medical School, 1-1-50, Fukushima-ku, Osaka 553, Japan [T. E., M. I., N. T., O. T.J

ABSTRACT

The role of cellular oncogenes in the development of epithelial tumorsof the human female reproductive tract has not previously been extensively studied. DNAs isolated from ten human uterine, 13 ovarian, andfour cervical neoplasms and from three cell lines derived from endometrialadenocarcinoma were investigated by dot blot hybridization after polym-erase chain reaction amplification of ras gene sequences and in somecases by NIH 3T3 transfection. Transforming activity was found in twoof nine endometrial adenocarcinomas, but none of seven ovarian carcinomas and none of four cervical carcinomas showed transforming activity.K-ra.vsequences with a GGT—»GATmutation in codon 12 were demonstrated in both transformants derived from endometrial carcinoma, h-ravcodon 12 mutations were similarly detected in six of 13 endometrialcarcinomas (one GAT and GCT, one GTT and GCT, two GAT, two GTT)and two of 13 ovarian tumors (GAT and GCT, GAT), both mucinousadenocarcinomas. Point mutation of k-rav in codon 12 is thus comparablyfrequent in uterine endometrial carcinomas and in colorectal carcinomasand may have similar significance as an event that contributes to progression of these tumors. Cervical carcinomas and ovarian tumors ingeneral, with the possible exception of mucinous adenocarcinoma of theovary, do not appear to have this characteristic.

INTRODUCTION

DNA-mediated gene transfer techniques have enabled thedetection of activated transforming genes in animal and humanneoplasia (1). A large proportion of oncogenes detected by thisassay are H-ras (2, 3), K-ras (4, 5), and N-ras (6, 7), and about10 to 30% of human tumors are revealed to contain activatedras genes by this assay ( 1-7). These ras genes are known to beactivated either by a point mutation in codon 12, 13, or 61 orby amplification of the wild-type alíeles(1). ras gene mutationscan be detected by selective oligonucleotide probe hybridization(8-10) or, for some types of mutation, by restriction fragmentlength polymorphism (11-13).

PCR2 is a technique to enzymatically amplify genomic DNAsequences by more than 105-fold (14). In combination with

selective oligonucleotide hybridization, detection of ras genemutations has been simplified (15). About 50% of humancolorectal carcinomas are revealed to contain ras mutations bythis method (16). In studies of RNase A mismatch cleavage ofPCR fragments, it was found that most human exocrine pancreatic carcinomas have K-ras mutations (17).

Malignant tumors of the female reproductive tract are common. Endometrial carcinomas represent 13% of all malignancies in women in the United States, ovarian tumors 6%, andother reproductive tract malignancies 2% to 3% (18). Therelationship of oncogene activation to certain gynecologicalmalignancies has been examined (19-21). Human papillomavirus type 16 and type 18 infections are associated with squa-

Received 12/21/89; accepted 7/5/90.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.

1To whom requests for reprints should be addressed.2The abbreviations used are: PCR. polymerase chain reaction: SDS, sodium

dodecyl sulfate; DMEM, Dulbecco's modified Eagle's medium; SSC, standardsaline citrate (0.15 M sodium chloride:0.015 M sodium citrate. pH 7.4); ffu, focus-forming units; IMI', low malignant potential.

mous cell carcinoma of the uterine cervix (22), and pointmutations in codon 12 of H-ras are found in 20 to 30% of thesetumors at Stages III and IV (23). K-ras activation has also beenreported for ovarian carcinomas (24, 25).

However, there are no systematic studies of the presence ofoncogenes in different classes of gynecological tumors, andespecially little is known about endometrial adenocarcinomas.We have therefore evaluated a series of human gynecologicalneoplasms by NIH 3T3 transfection assay and selective oligonucleotide hybridization of PCR-amplified fragments from tumor and tumor cell line DNAs.

MATERIALS AND METHODS

Tissue Samples. Tumors used in this study were from patients whowere admitted into either the Department of Obstetrics and Gynecologyat the Osaka University Hospital or the public hospitals in Osaka,Japan. No initial anticancer chemotherapy or radiation therapy wasperformed prior to tumor excision. Tumors were surgically removedand sampled for histopathological diagnosis, and remainders werefrozen. Histological classification of tumors was carried out accordingto the WHO international system (26). They included 10 endometrialadenocarcinomas, 4 squamous carcinomas of the uterine cervix, onemalignant melanoma of the vagina, and 13 ovarian tumors. Among theovarian tumors were 10 common epithelial tumors, one sex cord-stromal tumor, and 2 germ cell tumors. The clinical stage of the diseaseswas also established according to the International Federation of Gynecology and Obstetrics staging system. For negative controls, normalterm placentas and normal myometrium from patients with nonneo-plastic conditions, were also frozen.

Endometrial Cancer Cell Lines. Three established endometrial cancercell lines, HEC (27), IK-90 (28), and HHUA (29), were cultured inDulbecco's modified Eagle's medium supplemented with 10% fetal

bovine serum. To confirm the tumorigenicity of each line, cells wereinjected into athymic nu/nu mice (10* cells/injection, 2 injections/mouse). Two subclones of normal NIH 3T3 cells, NIH 3T3-C and NIH3T3-W, which were kindly provided by Dr. Yasuo Kokai, and two linesof NIH 3T3 transformants, which were derived from mouse liver tumorsand contained amplified H-ras sequences with a CAÕ—»CTAmutation(1-me) or a CAÕ—»AAAmutation (4-mc) in codon 61, were also injectedas controls.

DNA Preparation. For preparation of high-molecular-weight DNAfrom tissues, frozen samples were thawed and finely minced with sharpscissors. Minced tissues were lysed with buffer containing 0.5% SDS,0.15 M NaCl, 2 mM EDTA, and 10 m\i Tris-HCl (pH 7.8) and digestedwith Pronase at 400 ^g/ml at 37°Cfor 16 h. DNA was isolated by

successive extractions with 1 volume of phenol, twice; 1 volume ofphenol/chloroform/isoamyl alcohol (25/24/1); and I volume of chlo-roform/isoamyl alcohol (24/1), followed by precipitation with 2 volumes of ethanol (30). Ten ^g of DNA were electrophoresed on 0.7%agarose gels, the gels were stained with ethidium bromide, and theaverage molecular weight of the DNA was estimated. Some tumorswere necrotic, and high-molecular-weight DNA (>20 kilobases) wasnot obtained. Only preparations in excess of 20-kilobase pairs in averagelength were used for transfection assays.

Transfection Assays. DNA transfection analysis was performed bythe calcium phosphate coprecipitation method (30, 31). Approximately20 ftg of precipitated high-molecular-weight DNA were added to a 10-cm culture dish in which 1.5 x IO5 NIH 3T3 cells had been seeded inDMEM supplemented with 10% heat-inactivated calf serum 24 h prior

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K-ros ACTIVATION OF HUMAN FEMALE REPRODUCTIVE TRACT NEOPLASMS

to transfection. After 22 h, DNA was removed and fresh DMEM with5% calf serum was added. DNA from the transformant 3T3 cell line 1-mC was transfected as a positive control in each series of experiments.Cultures were maintained with medium changes every 2 to 3 days.Transformed foci were scored 14 to 21 days after transfection. Transformed cells from colonies were isolated and transferred to semisolidmedium (bottom layer, 0.5% agar; top layer, 0.3% agar), and individualcolonies were selected, expanded in monolayer culture, and harvestedfor extraction of DNA.

Southern Blot Analysis. Ten ^g of DNA were digested with theappropriate restriction enzyme for 2 h as recommended by the supplier(Bethesda Research Laboratories, Gaithersburg, MD) and fractionatedon a 0.7% agarose gel. DNA was then transferred to a nylon filter(Micron Separations, Inc., Westboro, MA). Probes for H-ras or N-ras(Lofstrand Labs., Limited, Gaithersburg, MD) and for K-ras (OncogeneScience, Inc., Manhasset, NY) were 32P-labeled by nick translation. Theprobe for Alu sequences (Oncor, Inc., Gaithersburg, MD) was 32P

labeled by primer extension. Filters were prehybridized with 5x SSC,lOx Denhardt's solution, 0.05 M Na2HPO4/NaH2PO4, 50 Mg/ml ofdenatured salmon sperm DNA, and 1% SDS at 65°Covernight. The32P-labeled probe was then added to a final activity of IO7cpm/ml andhybridized overnight at 65"C. The filter was washed twice in 3x SSC

and 0.5% SDS at room temperature for 10 min and O.lx SSC and 1%SDS at 60°Cfor 60 min. Finally, the filters were autoradiographed at-70°C(32).

Polymerase Chain Reaction. PCR was performed to generate amplified fragments surrounding K-ras codon 12. The primers used were 5'-

CATGTTCTAATATAGTCACA-3' (A) and 3'-GTTATCTCCATT-TAGAACAA-5' (B). Primer A hybridized to the noncoding DNAstrand from positions -53 to -34, and Primer B hybridized to the

coding strand from positions +104 to +123 (17). Each was specific forthe human, and distinct from the mouse, K ™,vgene. Reaction mixtureswith 2 jig of template DNA were denatured initially for 5 min at 100'C.Then Taq polymerase was added. One cycle consisted of 1 min at 94°C(denaturation), 2 min at 50°C(annealing), and 3 min at 72°C(elonga

tion). A total of 35 cycles were performed. PCR was also performed togenerate amplified fragments surrounding the codon 12 of H-ras usingpreviously published primer sequences (33). Positive controls for dotblot hybridization were also generated by PCR, using a mutation-specific probe as one of the primers.

Analysis of Amplified DNA Fragments by Dot Blot Hybridization.Aliquots of amplified DNA incubation mixtures (5 M!)were blottedonto a nylon filter (0.2 firn: Biodyne). The filter was hybridized for 2 hwith Sx SSPE, 0.1% SDS, lOx Denhardt's solution, and 500 ^g/ml ofdenatured salmon sperm DNA at 59°C.A 20-mer antisense 32P end-

labeled probe surrounding codon 12 of K-ras (34) was added to theprehybridization buffer (5 x 10' cpm/ml, 1 x IO6 cpm/ng) togetherwith unlabeled wild-type probes (5x labeled quantities) to decreasenonspecific binding. The sequence of the wild-type probe was 5'-CCTACGCCACCAGCTCCAAC-3'. After hybridization for 16 h, fil

ters were washed twice in 6x SSC at room temperature for 10 min,once with 6x SSC at 59°Cfor 30 min, and finally with 2x SSC and1.0% SDS at 65°Cfor 5 min. The filters were exposed to X-ray film at-70°Cfor4tolOh.

Table 1 DIVAsused for detection of point mutation in K-ras

DNAno."13323.1471215181932202122Age(yr)38555556625252565862HECHHUAIK-90OriginEndometriumEndometriumEndometriumEndometriumEndometriumEndometriumEndometriumEndometriumEndometriumEndometriumEndometriumEndometriumEndometriumHistologySAdenocarcinoma

G3AdenocarcinomaGlAdenocarcinoma

G1AdenocarcinomaG1AdenocarcinomaG1Adenocarcinoma

G3AdenocarcinomaG1Adenocarcinoma

G2AdenocarcinomaG1Adenocarcinoma

GlAdenocarcinomacelllineAdenocarcinomacelllineAdenocarcinomacell lineNIH

3T3transfection

tage assay+(K-raj)+(K-ras)ND*ND——ND——ND—K-roscodon12GGT-*GAT

andGCTGGT-»GATGGT-.GTTGGT—GTT

andGCTwwWwwtwtGGT-.GATGGT—GTTwt

II

2293137

368

1013

NI Endometrium Stremai sarcoma

68624151685662NI52CervixCervixCervixCervixMyometriumMyometriumMyometriumMyometriumMyometriumSquamoiSquamo!SquamoiSquamoiNormalNormalNormalNormalNormal

NI

III1

ND

60 Vagina Malignant melanoma

NDNDND

ND

wtwtwtwt

wtwtwtwtwt

wt

5362791728302425261614354638NI72622960593378281821OvaryOvaryOvaryOvaryOvaryOvaryOvaryOvaryOvaryOvaryOvaryOvaryOvaryMucinousadenocarcinomaMucinousadenocarcinoma(LMP)Mucinous

adenocarcinomaSerousadenocarcinomaSerousadenocarcinomaSerousadenocarcinomaSerousadenocarcinomaClear

cellcarcinomaEndometrioidcarcinomaEndometrioidcarcinomaGranulosa

celltumorEmbryonalcarcinomaImmature

teratomaIIIIIIIIiniinuiniiiiND—ND———NDNDND—ND——GGT-^JAT

andGCTGGT-<;ATwtwtwtwtwtwtwtwtwtwtwt

" DNA number is identical to the DNA number in Fig. 2.* ND, not done; wt, wild type; NI, not identified; +, transformed foci were observed; —,no transformed foci were observed.

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K-ras ACTIVATION OF HUMAN FEMALE REPRODUCTIVE TRACT NEOPLASMS

N N

23.1-

2.3-

A BFig. 1. Southern blot analysis of DNAs derived from transformants. DNAs

were digested with /mid. Southern blotted onto a nylon filter, and hybridizedwith a K-raj-specific probe (Fig. \A) or with the probe specific for Alu sequences(Fig. \B). Lane N, NIH 3T3 cells; Lanes a and b. transformants derived fromendometrial adenocarcinoma from DNA No. 1 (Lane a) and from DNA No. 33(Laneb).

RESULTS

Transforming Genes. To detect transforming genes in thegynecological tumors, DNA transfection analysis was performed by the calcium phosphate coprecipitation method usingNIH 3T3 cells. As shown in Table 1, DNA from 2 of 9endometrial carcinomas had transforming ability. Transformation efficiencies of these DNAs, which were calculated asthe number of ffu/^g of DNA transfected, were 0.006 for DNANo. 1 and 0.013 for DNA No. 33. A positive control, the NIH3T3 cell line 1-mC transformed by H-ras, consistently yieldedfoci (0.10 to 0.15 ffu/^g) in each series of experiments.

To confirm that transformation of NIH 3T3 cells was associated with uptake of a transforming gene of human origin,DNA from transformants was examined for Alu sequences bySouthern blot analysis. As shown in Fig. IB, both transformantscontained Alu sequences. DNAs were also digested with arestriction enzyme and hybridized with H-ras-, K-ras-, or N-ras-specific probes. fcoRI-digested DNA from transformantswhich hybridized with the K-ras probe showed an extra 3.0-kilobase band, well separated from endogenous mouse sequences and identical in size to that expected for a K-ras geneof human origin, which was not detected in DNA from untrans-fected NIH 3T3 cells (Fig. \A); however, DNA hybridized withH-ras or N-ras did not have extra bands derived from humanDNA (data not shown). These data suggest that the activatedoncogenes in the transformants are K-ras derived from thehuman endometrial carcinomas. To characterize the type ofmutation, PCR was performed to generate amplified fragmentssurrounding codon 12 of K-ras. PCR reaction mixtures were

dot blotted on nylon filters and hybridized with mutation-specific oligonucleotide probes. As shown in Fig. 2, both transformants contained a GOT—»GATtransition mutation in codon12ofK-ras.

Detection of Point Mutation in Tumor DNA. To confirm thatGGT—»GATtransition mutations in the NIH 3T3 transform-ants occurred in the original tumors and not during the transfection process (35, 36), PCR was performed using DNA fromthe original tumor as a template. The reaction mixtures weredot blotted on nylon filters, and filters were hybridized withmutation-specific oligonucleotide probes. Our standard hybridization protocols can detect 1 to 3% of mutated ras alíeles(Fig.3). DNAs derived from both original tumors, which showedtransforming activity, contained the same GGT—»GATtransition mutation in codon 12 of K-ras as was observed in thecorresponding NIH 3T3 transformants (Fig. 4) (DNA Nos. 1and 33); moreover, one tumor also contained a GGT—»GCTtransversion mutation (DNA No. 1). In addition, DNAs fromone endometrial carcinoma cell line, HEC, and from one mutinous adenocarcinoma of the ovary, neither of which transformed NIH 3T3 cells, were found to contain GGT—»GATtransition mutations (DNA Nos. 20 and 36).

PCR was also performed using DNAs of which there wasinsufficient quantity for transfection assays. One endometrialcarcinoma (DNA No. 23) and one endometrial carcinoma cellline, HHUA (DNA No. 21), contained a GGT-.GTT transversion mutation. One endometrial carcinoma (DNA No. 34) hadboth GGT—»GCTand GGT—»GTTmutations. One mucinouscarcinoma of the ovary (DNA No. 5) contained both GGT—»GAT and GGT—»GCTmutations. No mutations were found atthe first position of codon 12 or at the second position of codon13 of K-ras. No GGC-> GAC or GGC-»AGC mutations incodon 12 of H-ras were detected in any of the 32 tumors tested(data not shown). No mutations in codon 12 of any of the ras

T T p p p'l '2 r1 r2 r3

lilil

B

D

K-ras Probe

codon 12

Wt (Gly)GGT

M1 (Asp)GAT

M2 (Val)GTT

M3 (Ala)GCT

Fig. 2. Dot blot hybridization with the mutation-specific probe for codon 12of K-ras. PCR was performed to generate amplified fragments surrounding codon12 of K-ras using genomic DNA from transformants derived from endometrialcarcinoma DNAs No. I (dot 7",) and No. 33 (dot 7"j) as templates. AmplifiedDNAs were blotted onto nylon filters and hybridized with wild-type oligonucleotide probe (A) and mutation-specific oligonucleotide probes (B to 0). From P¡to P4 are positive controls for GGT (/•,),GAT (/>,), GTT (P,), and GCT (/>„).

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K-TO.ÕACTIVATION OF HUMAN FEMALE REPRODUCTIVE TRACT NEOPLASMS

genes were observed in 6 samples of nonneoplastic myometriumor in 2 normal full-term placentas.

To confirm that the two endometrial carcinomas and onemucinous adenocarcinoma of the ovary that contained twodifferent mutations at the second position of codon 12 of K-raswere not due to nonspecific binding of the probes to the mismatched DNA fragments, we performed the hybridization inthe presence of non--'2P-labeled wild-type probe at 5x the

concentration of the labeled probe (Fig. 5). No signals wereblocked by adding the unlabeled probe for wild-type togetherwith the probes for mutated alíeles(Fig. 5^4). However, signalswere significantly blocked by an excess of unlabeled probeidentical in sequence to the labeled probe (Fig. 5B). Theseresults indicate that these signals are not due to nonspecificbinding of the probe.

Tumorigenicity of Endometrial Carcinoma Cell Lines. Threeendometrial cell lines, HEC, HHUA, and IK-90, were injectedinto athymic nu/nu mice (IO6 cells/injection, 2 injections/

mouse) (Table 2). HEC and HHUA cells, both of which contained activating mutations in codon 12 of K-ras, grew intovisible tumors (4 to 5 mm in diameter) 19 days and 5 days afterinjection, respectively, while IK-90 cells which did not containa codon 12 mutation had produced no tumors after 47 days.The transformed NIH 3T3 cell lines 1-mc and 4-mc eachdeveloped visible tumors in nu/nu mice 3 days after injection;however, two subclones of normal NIH 3T3 cells, NIH 3T3-wand NIH 3T3-C, had not produced visible tumors by 110 daysafter injection.

DISCUSSION

Detection of oncogene activation in human and experimentaltumors by NIH 3T3 cell transfection assay is well established(1), but most of the oncogenes detected by this assay are

GGT GTT

h(0) a (1)

b (1/2)

C (1/4)

d (1/8)

e (1/16)

f (1/32)

9 (1/64)

12345678

9 10 11 12 13 14 15 16

17 18 19 20 21 22 23 24

25 26 27 28 29 30 31 32

33 34 35 36 37 N, N?

N D D D D D Di "i r0 r-, r A r»- r f.

K-ras Probe

codon 12

B

Wt (Gly)

GGT

M1 (Asp)

GAT

M2 (Val)GTT

M3 (Ala)

GCT

Fig. 4. Dot blot hybridization with a mutation-specific probe for codon 12 ofK-ras. PCR was performed to generate amplified fragments surrounding codon12 of K-ras using purified gcnomic DNA. Amplified DNAs were blotted ontonylon filters and hybridized with oligonucleotide probes containing wild-type I IIor mutated sequences (B to lì).For (B to D). the unlabeled wild-type probe (5xthe quantity of the labeled probe) was also added to the hybridization buffer.DNA numbers are identical to the numbers in Table 1: endometrial carcinomas(Nos. 1,7, 12, 15, 18, 19. 20, 21, 22, 23. 32. 33, and 34); endometrial stromalsarcoma (No. 11); cervical carcinomas (Nos. 2, 29, 31. and 37); normal myometrium (Nos. 3, 6, 8, 10, 13. and A1,): normal term placenta (Nos. NI and N,);

malignant melanoma in the vagina (No. 4); and ovarian tumors (Nos. 5, 9. 14,16, 17. 24, 25. 26, 27, 28, 30, 35, and 37). From P, to Pt are positive controlsfor GGT (/>,), GAT (P2), GTT (/>,), ACT (P.), TGT </>,). and GCT (/>.).

Fig. 3. Sensitivity of dot blot analysis of PCR-amplified DNA. PCR wasperformed to generate amplified fragments surrounding codon 12 of K-ras usingDNA from normal placenta and the HHUA cell line that contains a mutated K-ra.v alíele(codon 12, GGT—»GTT).A series of 2-fold dilutions of amplifiedHHUA DNA was made by adding amplified DNA from normal human placenta.Samples (5 jjl) were blotted onto nylon filters and hybridized with the wild-typeprobe (.-I) and the probe for GGT—«GTTmutation (B). For B, the unlabeledprobe for wild-type (5x quantity of the labeled probe) was also added in hybridization buffer, a. undiluted HHUA; b. '/i dilution; c. Vf. d. Vt; e, '/¡f.f, VSKg. '/<*'.

h, undiluted placenta.

members of the ras gene family. The assay has also permitteddetection of other oncogenes, including lea (37), B-lym (38),oncD (39), met (40), and neu (41). Since larger genes are morelikely to be inactivated by shearing during DNA preparationand are less likely to be taken up intact by 3T3 cells, theefficiency of the assay is dependent on the size of the gene tobe transfected. In rat schwannomas induced by a single trans-placental exposure to /V-nitrosoethylurea, T—»Atransversion in

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K-ras ACTIVATION OF HUMAN FEMALE REPRODUCTIVE TRACT NEOPLASMS

(A) (B)1 2345678 32P-labeled 12345678

r, r. n Probes n P3 P4

1. GAT

Fig. 5. Blocking assay of dot blot hybridization. Ten n\ of PCR-amplified fragmentswhich showed point mutation in Fig. 4 (DNANos. 1, 33, 23, 34, 20, 21, 5, and 36) were dotblotted onto nylon filters. Filters were thenhybridized with "P-labeled probe for a mutant

sequence (GAT. GCT, or GTT) in the presenceof a 5-fold excess of unlabeled probes for bothwild-type (GGT) and for a different mutation(A), or a 5-fold excess of both unlabeled wild-type and unlabeled probe identical to the same"P-labeled mutant sequence (B). From P, toPt are positive controls for GGT (/>,). GAT(P2), GCT (Pj), and GTT (P4).

(cold probes: GGT,GCT) (cold probes: GGT,GAT)

2. GCT

(cold probes: GGT,GAT) (cold probes: GGT.GCT)

3. GTT

(cold probes: GGT,GAT) (cold probes: GGT,GTT)

Table 2 Tumorigenidty of endometrial adenocarcinoma cell lines in athymicnu/nu mice

The cells were injected into athymic nu/nu mice (IO6 cells/injection, 2 injections/mouse). HEC, HHUA, and IK-90 are endometrial carcinoma cell lines, and1-mc and 4-mc are NIH 3T3 transformants derived from mouse liver tumorswhich contain amplified H-ras sequences with codon 61 mutations. Maximumgrowth means that the mice are euthanized because tumors had grown so largethat ulcérationsor erosions appeared on the surface of the tumors.

Cell line

Visible growth Maximum growth(4-5 mm in (>10 mm indiameter) diameter) ras activation

Endometrial adenocarcinoma

HECHHUAIK-90

1-mc4-mc

NIH 3T3-CNIH 3T3-W

19 days 38 days5 days 44 daysNo growth after 44 days

NIH 3T3 transformants

3 days 13 days3 days 9 days

Normal NIH 3T3

No growth after 110 daysNo growth after 110 days

K-ras 12 (GGT-.GAT)K-ras 12(GGT-^iTT)

Not detected

H-ras 61 (CAA-.CTA)H-rai 61 (CAA-.AAA)

Not detectedNot detected

the transmembrane domain of neu (a larger gene than K-ras)was demonstrated in 12 of 13 tumors by selective oligonucleo-tide hybridization; however, only 6 of 12 tumors with T—»Atransversion showed transforming ability in the NIH 3T3 trans-fection assay (42). Therefore, once the oncogenes involved inthe tumors are identified by NIH 3T3 transfection assay, alternative methods should be used to quantify the frequency ofoncogene activation, ras genes can be activated by point mutation in codon 12, 13, or 61 ( 1). These mutations can be detectedby selective oligonucleotide hybridization (8-10) or, in somecases, by restriction fragment length polymorphisms for certainmutations that introduce a new restriction site (11-13). Byselective oligonucleotide probe hybridization of PCR-amplified

fragments, point mutations in ras genes can be detected easily(15).

We tested gynecological tumors from Japanese patients bythe NIH 3T3 transfection assay and found that only 2 of 9endometrial carcinomas yielded DNA that transformed NIH3T3 cells. The transformants contained transforming K-rassequences with a GGT—>GATtransition mutation in codon 12.From these results, we inferred that point mutations in codon12 of K-ras play some role in endometrial carcinomas. Byselective oligonucleotide probe hybridization after PCR, whichis one of the most sensitive methods now available to detectpoint mutation, we demonstrated that 6 of 13 endometrialcarcinomas (46%) had point mutations in codon 12 of K-ras(Table 3), which is about the same frequency as found in humancolorectal tumors (34). These findings suggest that point mutations in codon 12 of K-ras are significant events in the etiologyof adenocarcinoma of the endometrium.

Four Stage I cervical squamous cell carcinomas did not showtransforming ability or contain point mutations in codon 12 ofeither K-ras or H-ras. These findings are consistent with thoseof Riou et al. (23) in which codon 12 mutations in H-ras werefound in 4 of 20 Stage III cervical carcinomas and in 3 of 9 inStage IV, but in tumor tissue from only one patient of 21 withStage I and from none of 26 with Stage II disease.

With regard to oncogene activation in human ovarian tumors,Feig et al. (24) reported that one of five ascites fluids collectedfrom patients with serous cystadenocarcinoma of the ovary hadtransforming K-ras sequences. Haas et al. (25) reported thatone of 18 ovarian carcinomas had transforming N-ras sequences, though the type of mutation in N-ras was not characterized. Boltz et al. (43) reported infrequent K-ras amplification.

Although the ovarian tumors we studied by the NIH 3T3transfection assay did not show any transforming activity, preparations of PCR-amplified tumor DNA sequences from 2 of

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Table 3 K-ras activation in gynecological tumors

Origin and histologyFrequency

(K-rajcodon 12 mutation)

UterusCorpus

Endometrial adenocarcinoma°

Endometrial stromal sarcomaCervix

Squamous cell carcinoma

OvaryCommon epithelial tumors

Mucinous adenocarcinomaSerous adenocarcinomaClear cell carcinomaEndometrioid adenocarcinoma

Sex cord-stromal cell tumorsGranulosa cell tumor

Germ cell tumorsEmbryonal carcinomaImmature teratoma

VaginaMalignant melanoma

6/13 (2 GAT, 2 GTT, 1 GATand GCT, 1 GTT and GCT)

0/1

0/4

2/3 (1 GAT and GCT, 1 GAT*)

0/40/10/2

0/1

0/10/1

0/1" Includes three endometrial adenocarcinoma cell lines.* One tumor, diagnosed as low malignancy potential, contained K-ras activated

bycodon 12 mutation.

the 13 tumors contained K-ras codon 12 mutations. These twotumors were mucinous adenocarcinomas; therefore, 2 of 3mucinous adenocarcinomas tested (67%) had a point mutationin codon 12 of K-ras, suggesting possible specific involvementof the K-ras mutation in this type of ovarian tumor. This differsfrom the findings of Van't Veer et al. (44) who reported no ras

mutations in 33 ovarian tumors including 3 mucinous carcinomas. Though they found strong hybridization of some tumorDNAs with mutation-specific probes, they concluded that thesewere in all probability due to cross-hybridization. We couldovercome problems with nonspecific binding by adding unla-

beled probe for the wild type in hybridization mixtures. Moremucinous ovarian adenocarcinomas must be studied before anydefinitive conclusion can be drawn.

One of the mucinous adenocarcinomas with a K-ras mutation

was diagnosed as a tumor of LMP (borderline malignancy),which is characterized pathologically as tumors that have neo-

plastic epithelial cells, cellular clusters detached from sites oforigin, increased mitotic activity, and nuclear abnormalities,but lack obvious invasion of the supporting stroma. LMPtumors differ from carcinomas by the absence of destructiveinfiltrative growth. The 5- and 10-yr survival rates in patientswith LMP tumors are 93% and 91%, respectively, comparedwith 34% and 29% for patients with invasive carcinomas (45).It would be interesting to investigate whether K-ras activationis related to the rare cases of malignant progression of LMPtumors, since some reports indicate that ras activation canoccur as a late event in tumor progression. In human colorectaltumors, 58% of adenomas greater than 1 cm in diametercontained ras mutations, while only 9% smaller than 1 cmshowed similar ras mutations (16). The human PAI teratocar-cinoma cell line has no transforming ras mutations in earlypassage in nude mice; however, in later passages a G—>Atransition in codon 12 of the human N-ras locus is detectableand correlates positively with cell tumorigenicity in nude mice(46). Our data on tumorigenicity of three endometrial carcinoma cell lines in nude mice also support the correlationbetween K-ras activation and tumor aggressiveness.

ras mutations also have been found in benign adenomas ofhuman thyroid (47) and in the premalignant condition, myelo-

dysplasia (48), with almost the same frequency as in malignanttumors of these tissues (49, 50). In mice, benign papillomas ofthe skin (51) or even normal skin chronically exposed to 12-0-tetradecanoylphorbol-13-acetate (52) contained point mutations in codon 61 of H-ras. These data indicate that the rasmutation can also occur as an early event in the process ofcarcinogenesis. However, the findings of two different ras mutations in some of the tumors argue that ras mutations are notnecessarily initiating events in these tumors. Further study willbe necessary to define the role of ras gene activation in thesedifferent categories of gynecological tumors.

ACKNOWLEDGMENTS

We thank Dr. Hiroyuki Kuramoto for providing HEC cells. Dr.Isamu Ishiwata for HHUA cells, and Dr. Gregory S. Buzard for usefulexperimental recommendations.

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1990;50:6139-6145. Cancer Res   Takayuki Enomoto, Masaki Inoue, Alan O. Perantoni, et al.   Reproductive Tract

Activation in Neoplasms of the Human FemalerasK-

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