Expression of the ras oncogene in gynecologic tumors

Nuovo, Cottral, and Richart

12. Nuovo Gj, Nuovo MA, Cottral S, et al. Histological correlates of clinically occult human papillomavirus infection ofthe uterine cervix. Amj Surg Patho11988; 12: 198-204. 13. Ferenczy A, Mitao M, Nagai N, et al. Latent papillomaviruses and recurring genital warts. N Engl j Med 1985;313:784-8. 14. Macnab jC, Walkinshaw SA, Cordiner jW, et al. Human papillomavirus in clinically and histologically normal tissue of patients with genital cancer. N Engl j Med 1986;15:1052-8.

February 1989 Am J Obstet Gynecol

15. Lorincz AT, Temple CF. Patterson jA, et al. Correlation of cellular atypia and human papillomavirus DNA sequences in exfoliated cells of the uterine cervix. Obstet Gynecol 1986;68:508-12. 16. DeVilliers EM, Schneider A, Miklaw H, et al. Human papilloma virus infections in women with and without abnormal cytologies. Lancet 1987;1:703-6.

Expression of the ras oncogene in gynecologic tumors Timothy J. O'Brien, PhD, Gary A. Bannon, PhD, David S. Bard, MD, James W. Hardin, PhD, and J. Gerald Quirk, Jr., MD, PhD Little Rock, Arkansas Control of oncogene expression has been shown to be a coordinated regulatory mechanism in normal growth and development. Overt expression of these genes also has been noted in transformed or neoplastic cell types. The ras family of oncogenes has been shown to be particularly evident among genes expressed in malignant tissues. We provide evidence, using ribonucleic acid dot analysis and Western blot analysis of gynecologic tumor extracts, that ras expression may be a common occurrence in these malignancies. Furthermore, the ras-related peptides can be detected in sera of some patients with tumors. (AM J QBSTET GVNECOL 1989;160:344-52.)

Key words: Oncogene, p21, ras peptide, ras-related peptides, RNA, dot blot, Western blot, gynecologic tumors Several tumor-associated proteins measurable in blood can be used to monitor tumor regression, progression, or recurrence. I For patients with gynecologic malignancies, two such proteins are useful indicators of disease status; they are human chorionic gonadotropin (hCG) for trophoblastic disease and CA 125 for serous ovarian cystadenocarcinomas!' 3 Unfortunately, there are few monitoring systems for most patients with gynecologic cancer. Recent investigations offer the promise that monitoring the expression of oncogenes may provide a new approach to evaluating patients with solid malignant tumors:' 5 Cellular oncogenes are expressed as an intrinsic part of the transformed or neoplastic phenotype! This group of genes, numbering about 40, plays a determining role in normal cellular development and difFrom the Departments of ObstetTlcs and G.vnecology, BlOchnn1stry and Molecular Biology, and Medicme, Umversit,v of Arkansasfor Medleal SCIences. Supported in part by a grant from the University of Arkansas for Medical SCIences FoundatIOn Fund and National Institutes of Health Grant CA 40406. Receivedfor publication November 25, 1987; revlSedJune 1, 1988; accepted August 1, 1988. Reprint requests: Timothy J. O'Brien, Slot 518, UAMS, 4301 W. Markham, Little Rock, AR 72205.

344

ferentiation. 5 . 7 That oncogene expression is intrinsically associated with the neoplastic growth of human tumors is supported by the findings of Slamon et al.," who showed that the oncogenes myc and ras are commonly expressed in almost all of 54 malignant tumors examined. They also reported a high incidence of fos expression and a less frequent expression of fes, fms, src, myb, and abl in some subclasses of tumor. Of the gynecologic malignancies evaluated, seven of eight expressed myc, Ha-ras, and K-ras, as determined by messenger ribonucleic acid (mRNA) dot blot analysis. In addition, fos was highly expressed, and in some ovarian tumors fms expression also was noted. The protein products of several oncogenes have been identified in fixed or frozen sections of tumors removed at the time of surgery. Several recent reports described the presence of ras-encoded and ras-related peptides in tumor tissues, including primary and metastatic colorectal tumors, prostatic carcinomas, malignant and benign colonic tumors, and carcinomas of the breast."·'2 In colorectal carcinoma, p21 expression was found to be increased significantly in primary tumors as compared with normal tissues. 9 In prostatic carcinoma the amount of p21 correlated with lack of differentiation of the tumors. 10 In contrast, neither prostatic acid phos-

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phatase nor carcinoembryonic antigen correlated with tumor grade or malignant potential. In colonic cancers p21 was expressed at a higher level in malignant disease than in either benign or normal tissue with immunoperoxidase techniques. II Elevated ras expression also has been shown to correlate with lymph node metastasis in patients with breast cancer (unpublished data). As part of a continuing investigation designed to assess the clinical utility of assaying oncogene peptide products in association with gynecologic malignancies, we examined expression of the ras oncogenes in patients with genital tract cancer. We chose ras for our initial investigation because of the following: (1) The ras family of genes is well characterized; (2) the peptide products of these genes are known and have been se quenced; (3) monoclonal and polyclonal antibodies are available for detecting ras peptides; (4) the ras gene products have been detected in several kinds of malignant tissues. In this study we present evidence that ras is expressed in several gynecologic malignancies and that its peptide product, p21, can be found in sera of patients with gynecologic cancer. Material and methods

Tissues and sera. All tissues were obtained at the time of surgery and were placed immediately in icecold phosphate-buffered saline solution. They then were diced into 2 to 3 mm cubes and stored frozen at - 70° C until needed. Blood samples were collected in red-top tubes and allowed to clot. The separated serum was stored frozen at - 70° C until needed. mRNA dot blots. RNA was extracted by the guanidine isothiocyanate method of Chirgwin et aI., 12 layered over 6.7 mollL cesium chloride, and centrifuged in a Beckman SW 490 rotor at 35,000 rpm for 16 hours. The resultant RNA pellet was dissolved in 10 mmollL Tris (pH 7.5), 1 mmollL ethylenediaminetetraacetic acid, and 0.2% sodium dodecyl sulfate. After the addition of 0.25 mol/L sodium acetate the RNA was precipitated by the addition of two to three volumes of ethanol and stored at - 70° C under ethanol until needed. For mRNA purification, total RNA was dissolved in 20 mmol/L Tris (pH 8.0), 250 mmollL ethylenediaminetetraacetic acid, 75 mmol/L sodium chloride, and 0.5% sodium dodecyl sulfate, then heat shocked for 1.5 minutes at 90° C. The RNA was immediately cooled according to the method of Thomas. 13 An equal volume of 2 x binding buffer (1.0 mol/L sodium chloride, 20 mmol/L Tris [pH 8.0], and 0.2% N-lauroylsarcosine) was added and the sample loaded onto an oligo dt cellulose column (Collaborative Research), according to Avid and Leder.I< The column was washed with 1 x binding buffer until the optical density at 260 nm approached zero. The column was then eluted with 0.01 moll L Tris, pH 8.0, and 0.1 %

Oncogene ras in gynecologic tumors 345

N-lauroylsarcosine. mRNA was precipitated with two volumes ethanol after addition of sodium acetate to a final concentration of 0.3 moll L. The resultant mRNA pellet was stored under ethanol - 70° C until needed. Dot blotting was performed by transfer of RNA to nitrocellulose filters by use of 1 : 3 serial dilutions for five dilutions of a 10 fJ..g starting RNA load. Filters were washed and hybridized according to conditions described by Thomas." The c-H-ras clone used to probe dot blot filters is a 6.6 kb insert subcloned from a Charon 4A genomic clone ofthe H-ras-gene. This fragment was subcloned as a BamH I fragment into a BamH site of pBR322. The plasmid was propagated in the Escherichia coli K-12 derivative HB 10 1 and was labeled with phosphate 32-nucleotide triphosphates and deoxyribonucleic acid (DNA) polymerase I. Polyacrylamide gel electrophoresis, blotting, and probing. Tumor tissues were extracted in four volumes of buffer (4 mmol/L Tris hydrochloride, pH 6.8) per unit weight of tissue. Routinely, 200 mg of tissue in 0.8 ml of buffer was processed. Tissue was homogenized on a Tekmar microprobe homogenizer with three 5second bursts; homogenization was interrupted by cooling on ice for 2 minutes between bursts. Extracts were centrifuged at 10,000 g for 20 minutes; the supernatant was removed and divided into 200 fJ..l aliquots. Samples were reduced to dryness on a Speedvac lyophilizer. Samples were resuspended in 40 fJ..l sample buffer (62.5 mmol/L Tris [pH 6.8], 5% mercaptoethanol, 10% glycerol, and 3% sodium dodecyl sulfate) and boiled for 2 minutes before electrophoresis on 12% sodium dodecyl sulfate-polyacrylamide gels. Electrophoresis was carried out for 3 1/2 hours at 200 V according to Laemmli. 15 The gels were transblotted to nitrocellulose filters in Tris-glycine buffer (25 mmollL Tris [pH 8.3], 192 mmol/L glycine, 20% vol/vol methanol) overnight at 40 V. Unoccupied nitrocellulose binding sites were saturated by exposing the filters to "blotto" (5% Carnation powdered milk in phosphatebuffered saline solution with 0.05% azide)'6 at 4° C for 2 hours. Primary antibody diluted 1: 200 in blotto was incubated with the filters overnight at 4° C. Second antibody (goat antirabbit for the polYcional primary antibody or rabbit antimouse for the monoclonal primary antibody) was incubated at I: 1000 dilution (Cooper Biomedicals Co.) for 2 hours at 4° C. Filters were then rinsed twice in phosphate-buffered saline solution and exposed to protein A labeled with iodine 125 (106 cpm/ml) for 90 minutes at room temperature. Unbound ['25 1]-protein A was removed by rinsing the filter five times with phosphate-buffered saline solution. Filters were exposed to x-ray film overnight at -70° C, and the film was developed in an automatic film developing machine. Fast protein liquid chromatography. Serum samples

346 O'Brien et al.

February 1989 Am J Obstet GynecoJ

ugRNA

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Table I. Relative ras expression: Conversion of dot blot data with densitometer readings used to derive relative abundance of H-ras mRNA III 10 tissue extracts TIssue type Serous ovarian carcinoma Uterine sarcoma Serous ovarian carcinoma Serous ovarian carcinoma Endometrioid ovarian carcinoma Endometrial adenocarcinoma Serous ovarian carcinoma Normal myometrium Normal endometrium Normal myometrium

RelatIVf mRNA content

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were diluted from 100 to 500 fl.l with 20 mmoilL BisTris propane (pH 7.2), then passed over a mono Q anion exchange column (Pharmacia, Inc.), with a linear sodium chloride gradient (0 to 375 mmollL) used to elute the bound proteins. Fractions of 1 .ml were collected and 200 fl.l aliquots were dot blotted to nitrocellulose with a Schleiker and Schuell dot blot apparatus. Nitrocellulose filters were blocked with blotto and probed as described previously. When individual or pooled samples were subjected to a second dimension of analysis on sodium dodecyl sulfate-polyacrylamide gels, they were dialyzed against 4 mmollL Tris hydrochloride (pH 6.8) and lyophilized to dryness before resuspension in sodium dodecyl sulfate. Analysis of serum samples for ras-related peptides. Albumin was removed from serum samples for polyacrylamide gel electrophoresis to overcome excessive distortion of the gels caused by protein overloading. Serum (100 fl.l) was incubated with 0.5 ml of affigel blue-agarose (Biorad, Inc.) for 30 minutes at 4° C. Samples were then centrifuged at 2000 g for 10 minutes at 4° C, and the supernatant was removed and reduced

to dryness by lyophilization or equilibrated with sodium dodecyl sulfate sample buffer and electrophoresed directlyon 12% sodium dodecyl sulfate-polyacrylamide gels after the sample was boiled for 2 minutes. Blotting and probing of the filters were carried out as described. Immunoprecipitation of ras peptides. Normal tissues and benign or malignant tumor tissues were extracted with four volumes of buffer (4 mmol/L Trishydrochloride, pH 6.8) per unit weight of tissue as described. Aliquots of extract (200 J.Ll) were diluted with 300 fl.1 of albumin buffer (50 mmollL Trishydrochloride containing 0.5% bovine serum albumin, pH 67.8) and incubated with 2.5 J.Ll (I: 100 dilution) of monoclonal ras antibody overnight at 4° C. The following morning 200 fl.l of rabbit antimouse immunoglobulin, attached to agarose beads (Biorad, Inc.) diluted 1 : 20, was added to the incubation mix and incubated on a shaker for 2 hours at room temperature. The beads were removed by centrifugation at 2000 g for 10 minutes and washed twice with 1.5 ml of phosphatebuffered saline solution. The beads were then boiled in 100 fl.l of electrophoresis sodium dodecyl sulfate buffer, and 40 J.LI of each sample was electrophoresed as described above. After electrophoresis the peptides were transferred from the polyacrylamide gel to a nitrocellulose blot and probed with monoclonal ras antibody overnight as before. Antibody binding was localized with rabbit antimouse used as second antibody; it was diluted I: 1000 in blotto and incubated for 2 hours at 4° C. The blot was washed three times in phosphate-buffered saline solution and incubated with 10 ml [12'1]-protein A (10" cpm/ml) for 90 minutes at room temperature. After washing, the filter was exposed to x-ray film at - 70° C overnight.

Results c-H-ras RNA transcripts and p21 ras peptides in gynecologic tumors. To determine the level of expres-

Oncogene ras in gynecologic tumors

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347

P200

P21

2

3

1

2

30 P200

P21

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3

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Fig. 2. Analysis of gynecologic tumor extracts for p21 and related proteins by Western blotting with polyclonal anti-ras antibodies (A) and monoclonal anti-ras antibodies (8). Lane 1, endometrioid ovarian carcinoma; 2 to 4, serous ovarian carcinoma; 5, uterine sarcoma; 6, endometrial adenocarcinoma. C, Normal and benign tissue extracts. Left, Polyclonal ; nght. monoclonal. Lane 1, myometrium; 2, endometrium; 3, hyperplasia. D, Immunoprecipitation of normal tissue and benign and malignant tumor tissue extracts. Lanes 1 and 2, serous ovarian carcinoma; 3 and 4. leiomyomas; 5 and 6, myometrium; 7, endometrium.

sion of the H-ras gene in gynecologic neoplasms, we extracted RNA from a series of tumor tissues and neighboring normal tissues . With the dot blot technique, serial dilutions of RNA were made and probed with a radioactive c-H-ras DNA probe. The presence of c-ras mRNA is observed by the retention of the DNA probe on the filter, which when exposed to x-ray film is converted to an increased density of the dot. Fig. I shows a series of RNA extracts from seven malignant tumors (lanes I to 7) compared with RNA extracted from three normal tissues (lanes 8 to 10). This approach indicates that several malignant tumor extracts contain increased levels of H-ras mRNA. When data are converted numerically by use of densitometer readings, the results can be tabulated on a relative abundance scale of I to 4. As such, it is apparent that the uterine specimens (lane 2, the sarcoma, and lane 6, the endometrial adenocarcinoma) and the endometrioid carcinoma of the ovary (lane 5) all contain relatively high levels of H-ras mRKA (Table I). To confirm that the H-ras-encoded mRNA was translated in tumor tissues, six tumor extracts that contained H-ras-encoded mRNA together with two nor-

mal tissue extracts and one benign tissue extract were prepared for sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis. We found that the malignant tumor tissues also contained detectable amounts of the p2 I -encoded peptide product (Fig. 2, A and B). Whether the immunoblots were probed with a polyclonal antibody (Fig. 2, A) (which recognizes epitopes toward the amino terminal of the p21 molecule) or with a monoclonal antibody (which recognizes the amino acid sequence 96-118 located toward the carboxyl end of the molecule), p21 was detected (Fig. 2, B). The polyclonal antibody also recognized lower-molecular-weight species (18,000 to 14,000 daltons), which probably represent proteolytic products of the native ras-encoded peptide (Fig. 2, A). In addition, the polyclonal antibody also recognized ras-related peptides of higher molecular weight in all four of the malignant ovarian tumor extracts (lanes I to 4, Fig. 2, A). The uterine sarcoma extract contained a 180 kd species (lane 5, Fig. 2, A). The uterine sarcoma extract contained a 180 kd species (lane 5, Fig. 2. A) , and the endometrial carcinoma extract contained a 190 kd species (lane 6, Fig. 2, A). These ras-related species

348

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February 1989 Am J Obstet Gynecol

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and those recognized at approximately 45 kd (Fig. 2, A, lane 3) are probably products of genes with epitopes

or domains shared with p21. Further confirmation that ras-encoded p21 is present in malignant tumor extracts was obtained by probing gels with a monoclonal antibody raised against a highly conserved region in the p21 peptide" (Fig. 2, B). As with the polyclonal antibody, ras-related peptides of high molecular weight were recognized by the monoclonal antibody. Normal and benign tissue extracts probed with polyclonal or monoclonal antibody (Fig. 2, C) did not exhibit the same intensity of banding shown by the malignant tumor extracts, except for lane 3 (the endometrial hyperplasia extract), where monoclonal antibody did identify some p21. Some ras-related bands also were recognized in normal and benign extracts with both monoclonal and polyclonal antibodies. Immunoprecip-

itation of ras-related peptides from extracts of normal, benign, and malignant tumor tissues with monoclonal antibody followed by electrophoresis and Western blotting further demonstrated the presence of these peptides in malignant tumor tissues (Fig. 2, D, lanes I and 2). No immunoprecipitable peptides were detected in normal endometrium or myometrial extracts (Fig. 2, D, lanes 5 to 7). and a single peptide migrating at 21,000 daltons was detected in one (Fig. 2, D. lane 4) of two extracts of leiomyomas. ras Peptides and related peptides in patients' sera. The presence of both mRNA and p21 in gynecologic tumor extracts led us to assay blood from patients with malignant tumors for p21 and ras-related peptides. Sera from patients with malignant tumors and normal subjects were fractionated on the basis of charge on a mono Q anion exchange column with a fast protein

Oncogene ras in gynecologic tumors

Volume 160 I'\umber 2

349

P200

P21

CD'

GRADIENT ELUTION ........

Fig. 4. Two-dimensional analysis of ras and ras-related peptides. Fast protein liquid chromatography

with gradient elution followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of pool fractions. Western blotting, and ras detection with monoclonal anti-ras antibody. A, Tumor serum; B, normal serum.

liquid chromatography system. The serum was fractionated because preliminary studies demonstrated excessive nonspecific binding of antibodies to other serum proteins. Eluted fractions were dot blotted to nitrocellulose, incubated with polyclonal anti-H-ras antibodies, and probed with [125 I]_goat antirabbit immunoglobulin C. The antibody bound to the early eluting fractions of serum obtained from both patients with malignant tumors and normal subjects (Fig. 3, A). In contrast, the antibody bound to postalbumin eluates (fractions 26 to 34) only in sera obtained from tumorbearing patients (Fig. 3, B). These results suggest that ras-encoded or ras-related proteins can be detected in sera obtained from patients with malignant tumors. This approach, however, is time consuming, is cumbersome, and has limitations if multiple samples are to be evaluated. Furthermore. without knowledge of the molecular weight of the observed binding species. it is not possible to ascertain whether the antibody is binding to H-ras-encoded p21 or to other ras-related pro-

teins. To confirm that dot blot analysis of sera fractionated by fast protein liquid chromatography was recognizing p21 peptides, fast protein liquid chromatography elution fractions were pooled, dialyzed, concentrated. and electrophoresed on sodium dodecyl sulfate-containing polyacrylamide gels. Peptides thus separated by size were probed with the monoclonal antibody. Western blots confirmed the presence of the ras-encoded p21 peptide in serum from a patient with a malignant tumor (Fig. 4, A) and the absence of p21 in control serum (Fig. 4, B). To process multiple serum samples simultaneously, we electrophoresed them on 12.5% sodium dodecyl sulfate-polyacrylamide gels in an effort to detect the p21 protein. Proteins were transferred to nitrocellulose and probed with the polyclonal and monoclonal antibodies. Under direct electrophoresis conditions, high concentration of proteins in serum (particularly albumin) resulted in the overloading and distortion of antibody-reactive bands (data not shown). When al-

350 O'Brien et al.

February 1989 Am J Obstet Gynecol

NORMAL

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Fig. 5. Western blotting of serum samples from both normal patients and patients with tumors (A and B) after sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by ras detection with polydonal antibodies.

bumin was removed by affigel blue (see Material and methods section for details) before sodium dodecyl sulfate-polyacrylamide gel electrophoresis, p21 was readily detected by Western blot (Fig. 5) in 11 of 15 malignant tumor sera and none of two control sera shown here. Overall 23 of 35 malignant tumor sera and one of six normal sera were shown to contain p21 by Western blot analysis. Other prominent bands detected by the polyclonal antibodies migrate with apparent molecular weights of 14 kd (Fig. 5, A) and 55 kd (Fig. 5, A and B). Reactive bands at 50 and 30 kd likely represent immunoglobulin G heavy and light chains that react with the second antibody (goat anti rabbit immunoglobulin G) used in this assay. We compared the relative concentrations of ras-related proteins in sera from patients with malignant gynecologic tumor and from normal subjects. Both the 21 and 14 kd proteins

were more abundant in sera from patients with malignant tumors (Fig. 5, A, lanes 1 to 7, B, lanes 11 to 18) than in sera from normal subjects (Fig. 5, A, lanes 9 and 10). The ras-related band migrating at 55 kd was presen"t in variable amounts in both patients with malignant tumors and normal subjects. Control experiments with nonimmune mouse serum or immune sera to other peptides did not show localization of bands migrating at these molecular weights. From these data . we conclude that it is possible to detect the ras-encoded p21 peptide in the sera of patients with malignant gynecologic tumors.

Comment The H-ras-encoded peptide p21 and related proteins and peptide products can be detected in both tumor extracts and sera of some patients with gyne-

Volume 160 Number 2

cologic malignancies. When assayed in a semiquantitative fashion with polyclonal and monoclonal antibodies, sera obtained from these patients showed increased levels of p2l and ras-related proteins when compared with control samples. It is important that these serum samples also contain significant amounts of a 14 kd peptide (recognized by the polyclonal antibody), which previously has been shown to be a proteolytic product of p21 in transformed fibroblasts (Feramisco JR, personal communication). In a similar manner, extracts of tumor tissues probed with the polyclonal antibody clearly contain the p21 peptide and the 14 kd proteolytic product of p21. In addition, the polyclonal antibody also detected the presence of other ras-related proteins in these tumor tissues. With a monoclonal antibody '7 that recognizes a conserved sequence in p21 encoded by H-ras, K-ras, and N-ras, p21 was found both in patients' sera and in extracted tumor tissue. Both the polyclonal antibody and the monoclonal antibody recognized high-molecular-weight ras-related peptides in tumor tissue. A unique 25 kd peptide was prominent in Western blots of tumor tissues probed with the monoclonal antibody. In general, the presence of p21 peptides indicates the expression of one or all of the ras oncogenes in these tumor tissues. The polyclonal antibody recognizes antigenic epitopes toward the amino end of the p21 molecule and was raised to a mutant form of the H-ras p21. On the other hand the monoclonal antibody would be expected to recognize the p21 products of the N-ras, H-ras, and K-ras genes. Just recently a new member of the ras family of proto-oncogenes has been added, the so-called R-ras, or related ras, protooncogene. IS The peptide product of the R-ras gene contains an added 26 amino acid at the amino end of the molecule, resulting in an approximately 24 kd molecular weight peptide. Two amino acid sequences of the synthetic peptide made by Niman et al. '7 to generate the monoclonal antibody used here are conserved in the amino acid sequence of the R-ras peptide. Therefore it might be expected that the monoclonal antibody would recognize peptide products of the R-ras gene. The 25 kd band recognized by the monoclonal antibody may suggest the expression of this proto-oncogene. The presence of ras-encoded and ras-related peptides in sera of patients with malignancies provides evidence that the protein products of oncogenes may be of value in monitoring therapy in these patients. The ras peptides are not known to be secreted peptides but rather are membrane-associated and cytoplasmic oncogene products. The appearance of individual ras peptides in serum represents turnover of the membranes in transformed cells and also may reflect the expression of multiple ras genes in the transformed state. These genes are located on different chromo-

Oncogene ras in gynecologic tumors

351

somes, and their individual or collective role in malignancy has not been determined. Because individual p21 species may be distinguishable on the basis of their characteristic mRNA and net protein charge (and, for R-ras, size of the peptide product), a complete analysis of ras peptide products in gynecologic tumors will be undertaken. This approach may provide for a rational basis for the following: (1) identifying differences in ras expression in tumors of differing histologic types or grades, (2) identifying differences between primary and metastatic lesions, and (3) monitoring tumor behavior after treatment. Should individual p21 peptides be associated with a given prognosis, a management program with appropriate assays could be developed to address those needs. We express our appreciation to James R. Feramisco for providing polyclonal ras antibody and for his suggestions and contributions III preparing the manuscript. REFERENCES 1. Smith LH, Richard 01. Detection of malignant ovanan neoplasms: a review of the literature. III. Immunological detection and ovarian cancer-associated antigens. Obstet Gynecol Surv 1984;39:346-60. 2. O'Brien TJ. Trophoblastic disease monitormg. Clin Obstet Gynecol 1984;27:240-7. 3. Bast RC, Klug TL, St. John E, et at. A radioimmunoassay using a monoclonal antibody to monitor the course of epithelial ovarian cancer. N EnglJ Med 1983;309:883992. 4. Feramisco JR, Clark R, Wong G, et at. Transit revision of ras oncogene induced cell transformation by antibodies specific for amino acid 12 of ras protein. Nature 1985; 314:639-42. 5. Bishop JM. The molecular genetics of cancer. Science 1987;235:305-11. 6. Weinberg RA. The action of oncogenes in the cytoplasm and nucleus. Science 1985;230:770-6. 7. Hunter T. Oncogenes and proto-oncogenes: how do they differ? JNCI 1984;73:773-86. 8. Slamon DJ. deKernion JB, Verma 1M, Cline MJ. Expression of cellular oncogenes in human malignancies. Science 1984;224:256-62. 9. Gallick GE, Kurzrock P, Kloetzer WS, et at. Expression of p21 ras in fresh primary and metastatic human colorectal tumors. Proc Nat! Acad Sci USA 1985;82: 1795-9. 10. Viola MV, Fromowitz F, Oravez S, et at. Expression of ras oncogene in p21 in prostate cancer. N Engl .I Med 1986;314:133-7. 11. Thor A, Hand PH, Wunderlich D, et at. Monoclonal antibodies define differential ras gene expression in malignant and benign colonic diseases. Nature 1984;311: 562-5. 12. Chirgwin JM. Przybyla AE, MacDonald RJ, Rutter WJ. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 1979; 18:5294-9. 13. Thomas PS. Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Nat! Acad Sci USA 1980;77:5201-5. 14. Aviv H, Leder P. Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acid-cellulose. Proc Nat! Acad Sci 1972;69:1408-12. 15. Laemmli UK. Cleavage of structural proteins during the

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assembly of the head of bacteriophage T4. Nature 1970;227 :680-5. 16. Johnson DA. Gautsch JW. Sportsman JR, Elder JH. Improved technique utilizing nonfat dry milk for analysis of proteins and nucleic acids transferred to nitrocellulose. Gene Anal Tech 1984;1:3-8.

February 1989 Am J Obstet Gyneco1

17. Niman HL. Thompson AM. Yu A. et al. Anti-peptide antibodies detect oncogene-related proteins in urine. Biochemistry 1985;82:7924-8. 18. Lowe DG, Capon DJ. Delwart E. et al. Structure of the human and murine R-ras genes. novel genes closely related to ras proto-oncogenes. Cell 1987;48:137-46.

Association of human immunodeficiency virus - induced immunosuppression with human papillomavirus infection and cervical intraepithelial neoplasia Michelle J. Henry, BS: Michael W. Stanley, MD: Stephen Cruikshank, MD,"'c and Linda Carson, MDb.c Minneapolis, Minnesota Human papillomavirus infection plays an important causal role in cervical intraepithelial neoplasia and carcinoma. The rate of infection with human papillomavirus as well as the incidence of cervical intraepithelial neoplasia and carcinoma are increased in immunosuppressed patients. We report a possible association between infection with human immunodeficiency virus and cervical intraepithelial neoplasia with human papillomavirus infection. (AM J OasTET GVNECOL 1989;160:352-3.)

Key words: Human immunodeficiency virus. acquired immunodeficiency syndrome, human papillomavirus Historical observations have led to the recognition of several important epidemiologic factors that appear to playa role in the etiology of squamous cell carcinoma of the uterine cervix. The pattern of occurrence of carcinoma of the cervix is identical to that of a venereal disease, and the diagnosis of cervical intraepithelial neoplasia is often made based on cervical samples from women with a history of multiple sexual partners or husbands with penile cancer. Consequently, infectious agents, particularly viruses, have been implicated in cervical carcinogenesis. Cervical condylomas are the direct result of human papillomavirus infection, and deoxyribonucleic acid derived from the human papilloma virus family has been detected in human semen samples and biopsies of cervical intraepithelial neoplasia tissue as well as in samples of frankly invasive cervical carcinoma. The higher incidence of cervical carcinomas in

From the Departments of Pathologf and Obstetrics and Gynecology,' Hennepin County MedICal Center and DIVISIOn of Gynecologic Oncology, Department of Obstetrics and Gynecology.' Universtty of Minnesota. Received for publication May 9.1988; revised July 12.1988; acceptedAug. 1. 1988. Reprint requests: Michael W. Stanley, MD, Department of Pathology, 815. Hennepin County MedIcal Center, 701 Park Ave. South, Minneapolis. MN 55415.

352

women with renal allografts or after chemotherapy for Hodgkin's disease, as well as possible oncogenic transformation of rapidly proliferating (atypical) condylomata associated with pregnancy or diabetes mellitus, indicate that immunosuppressed women are at increased risk of human papillomavirus infection, cervical intraepithelial neoplasia, and cervical carcinoma. 1 We report a possible association of human papillomavirus (HIV)-induced immunosuppression with cervical condylomata and cervical intraepithelial neoplasia in four patients seen in Hennepin County Medical Center's Colposcopy Clinic (Table I). Material and methods

The four women comprising this study represent the entire adult female population with evidence of HIV infection who received their primary health care at Hennepin County Medical Center, Minneapolis, Minn. An initial Papanicolaou smear followed by col poscopic biopsy was obtained in the usual fashion. Antibodies to HIV were demonstrated by enzyme-linked immunosorbent assay and confirmed by Western blot analysis. Results and comment

As of Dec. 31, 1987 there were 670 HIV-positive patients (40 women) and 302 patients with acquired