Proc. NatL Acad. Sci. USA Vol. 79, pp. 3853-3857, June 1982 Medical Sciences Herpesvirus-specific RNA and protein in carcinoma of the uterine cervix (cloned DNA/in situ hybridization/immunoperoxidase staining/DNA-binding protein) JAMES K. MCDOUGALL*t, CHRISTOPHER P. CRUMt, CECILIA M. FENOGLIOt, LYNN C. GOLDSTEIN*, AND DENISE A. GALLOWAY* *Fred Hutchinson Cancer Research Center, 1124 Columbia Street, Seattle, Washington 98104; tDepartment of Pathology, University of Washington, Seattle, Washington 98105; and tColumbia University College of Physicians and Surgeons, 168th Street and Broadway, New York, New York 10032 Communicated by Earl P. Benditt, March 8, 1982 ABSTRACT Cloned probes of herpes simplex virus type 2 ment N of HSV-2 DNA (25, 26), which has been reported to DNA were used in cytological hybridization experiments to detect have transforming activity in rodent cells (27, 28). herpesvirus RNA transcripts in the neoplastic cells of tumors of Rodent cells transformed in vitro by HSV-2 have been re- the uterine cervix. Virus-specific RNA was shown to represent ported to express a number of HSV-specific antigens including transcription of limited regions of the genome, of which one is VP143 (29), ICP10 (30), thymidine kinase (31, 32), membrane known to code for a DNA-binding protein that can be found by glycoproteins gA/gB (33, 34), and CP-1 (35). We therefore ex- immunoperoxidase staining in the neoplastic cells of these tumors amined the HSV-2 transformants available in this laboratory in and has also been detected in cells transformed in vitro by this parallel with specimens of cervical carcinoma in an attempt to virus. find a protein universally present in cells transformed in vitro and in vivo The thesis that the continuum of disease leading to the devel- possibly by HSV-2. opment of uterine cervical carcinoma is associated with prior infection by herpes simplex virus type 2 (HSV-2) (1-5) has re- MATERIALS AND METHODS cently been tested at the molecular level by the use of in situ Cells and Viruses. The properties of the transformed cells cytological hybridization (6-9). The use of 3H-labeled viral used in this study have been described (11, 28, 36). Cells were DNAs as probes to detect virus-specific RNA (10, 11) in cervical cultured in an atmosphere of 10% C02/90% air in Dulbecco's tissue has resulted in general agreement, based on published modification of Eagle's medium (Bio-Rad) with 10% fetal calf reports, that at least 30% ofall cervical intraepithelial neoplasia serum (Reheis, Kankakee, IL) on plastic tissue culture plates (CIN) and cervical carcinoma tissues contain cells that bind a (Falcon). HSV-2 strain 333 was propagated on monolayers of HSV-2 [3H]DNA probe but not probes representing other viral BHK-21 cells infected at low multiplicity (0.01 plaque-forming DNAs, indicating the presence ofHSV-specific RNA (7-9). The unit per cell). Viral DNA was isolated by equilibrium gradient majority of these in situ hybridizations have been conducted centrifugation through sodium iodide gradients containing with whole genomic DNA as the probe. In this report we sum- ethidium bromide (37). marize our studies using cloned subgenomic fragments ofHSV- Human Uterine Cervical Tissues. Human cervical tissue was 2 DNA as hybridization probes to define regions of the virus obtained during colposcopic examination from patients who had genome from which the detected RNA species are transcribed been referred for the evaluation of abnormal Papanicolaou and correlate the findings with attempts to identify viral anti- smears. Biopsy specimens were taken from those areas most gen(s) expressed in cervical carcinoma cells. likely to contain cervical cancer precursors as identified by col- There have been numerous reports that antibodies to HSV- poscopy. The specimens were frozen in a cryostat, the diagnosis specific proteins are found more frequently, or at higher titer, was confirmed by examination of frozen sections, and perma- in women with CIN or cervical carcinoma than in controls nent diagnostic sections were prepared. The tissue remaining (12-14) and that HSV-specific antigens can be detected in the after the diagnostic evaluation was stored at -70'C until further neoplastic cells by immunofluorescent or other methods (15- sections were prepared. Benign cervical tissue from normal- 17). Among the abundance ofsometimes confusing data (18, 19), appearing cervical transformation zones was also obtained from two series ofrecent reports were ofinterest with respect to our hysterectomy specimens ofpatients whose uteri were removed in situ hybridization results. First, antiserum to the major HSV- for other reasons. These were snap-frozen and stored at -70'C. 2 binding protein (20) was shown to react with human cervical The cervical tissues were divided into three categories: (i) carcinoma cells in two separate studies (21, 22). The tumor an- benign (including squamous metaplasia), (ii) CIN (dysplasia and tigens of other DNA viruses have been shown, without excep- carcinoma in situ), and (iii) invasive cancer. The tissues obtained tion, to have DNA-binding properties (23), and a herpes simplex that did not include the transformation zone were also subjected tumor antigen might be expected to exhibit a similar function. to the hybridization studies despite the fact that cervical squa- Second, it was demonstrated that sera from patients with cervi- mous cell neoplasia generally does not arise in this region. The cal carcinoma precipitated two HSV-2 polypeptides (with mo- biopsies were interpreted as benign ifthe squamous epithelium lecular weights 38,000 and 118,000) more frequently than did matured in a normal fashion or if they contained the immature sera from controls (24). cells ofsquamous metaplasia at the surface or in the endocervi- The 38,000-dalton protein was ofinterest because it has been cal glands. The CIN lesions were classified by using the criteria shown that a protein of this size is encoded within Bgl II frag- advocated by Richart (38): CIN I, mild and moderate dysplasia; The publication costs ofthis article were defrayed in part by page charge Abbreviations: HSV-2, herpes simplex virus type 2; CIN, cervical in- payment. This article must therefore be hereby marked "advertise- traepithelial neoplasia; NaCl/Cit, standard saline citrate (0.15 M NaCl/ ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 0.015 M sodium citrate, pH 7). 3853 3854 Medical Sciences: McDougall et aL Proc. Nad Acad. Sci.' USA 79 (1982) 203 HinX B 304 Xba I D 203 201 102 201 0 - i ] * = E 305 301 401 EcoRI K r 18 r 510 J E EJ L--JI i I I ! I 0 1.0 FIG. 2. Distribution of HSV-2 DNA probes used for hybridization. ;yso..0 The solid boxes indicate positive results for HSV RNA. to remove all traces of ethanol, 10 A.l of probe was applied to each, and the drop-was covered with a washed, silicone-treated coverslip. Hybridization was carried out in 6 X standard saline E},,. t ' * citrate (NaCi/Cit; lx NaCi/Cit = 0.15 M NaCV0.015 M so- dium citrate) at 680C for 18 hr. After this period, the coverslips were removed and the slides were washed extensively in large volumes of 2x NaCl/Cit at 40C. Following dehydration in ethanol and under reduced pressure, the slides were dipped in Kodak NTB2 emulsion, diluted 1:1 with distilled water con- taining 1% glycerol, dried, and stored in light-tight boxes at 40C FIG. 1. In situ hybridization of 3H-labeled, nick-translated pDG304 were in Kodak DNA to frozen section of cervical intraepithelial neoplasia. (A) After for autoradiography. Preparations developed hybridization and staining with Giemsa. (x50.) (B) Higher magnifi- D19 and fixed in Kodafix. Cells and tissues were stained in 10% cation of a portion of the epithelium, showing localization of grains Giemsa, and the stained sections were examined for evidence over cells. (x300.) of silver grains. Cells with 10 or more grains were considered positive when the background level was 1-3 grains per cell. lines CIN II, moderate and severe dysplasia; and CIN III, severe Antibodies and Immunoprecipitation. Hybridoma cell the method dysplasia and carcinoma in situ. Invasive carcinoma was diag- producing monoclonal antibodies were isolated by nosed when the neoplastic cells penetrated through the base- of Kohler and Milstein (41). Spleen cells from C57BL/6 mice ment membrane and invaded the underlying stroma. infected with HSV-2 (333) were fused with BALB/c NSI my- Hybridization. The construction and characterization of the eloma cells by procedures described in detail elsewhere (41). recombinant clones of HSV-2 DNA in plasmid pBR322 have Culture supernatants were assayed for anti-HSV antibodies by an antibody-binding assay (42) utilizing "2I-labeled protein A been described (39). The introduction of 3H-labeled nucleo- HSV-2-specific tides into cloned DNA for use as hybridization probes was car- against HSV-2-infected cell antigens. Three in this study: one, antibody ried out as described (40). monoclonal antibodies were used a Frozen-sectioned biopsy tissues placed on glass slides were 6-A6, primarily precipitated 140,000-dalton protein together fixed and stored in ethanol at -20°C until required for hybrid- with less preference for a 38,000-dalton protein; conversely, ization. After the preparations were dried in vacuum desiccators antibody 6-Hll primarily reacted with a 38,000-dalton protein and less so with a 140,000-dalton protein (26). Antiserum (monospecific) to ICSP 11/12 was kindly provided by K. L. Table 1. In situ hybridization of subgenomic HSV-2 sequences to Powell (University of Leeds, England); anti-VP143 was pro- cervical intraepithelial and invasive neoplasia tissue samples vided by R. J. Courtney (University ofTennessee). % positive for HSV-specific RNA Immunoprecipitation reaction mixtures consisted of 100 ,ul Invasive [3H]DNA Map CIN carcinoma Non-neoplastic Table 2.