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Reaction of Antigens Isolated from Herpes Simplex Virus Transformed Cells with Sera of Squamous Cell Carcinoma Patients1

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Reaction of Antigens Isolated from Herpes Simplex Virus Transformed Cells with Sera of Squamous Cell Carcinoma Patients1 [CANCERRESEARCH36,4394-4401,December1976] Reaction of Antigens Isolated from Herpes Simplex Virus transformed Cells with Sera of Squamous Cell Carcinoma Patients1 Mary F. D. Notter and John J. Docherty2 Department of Microbiology and Cell Biology, The Pennsylvania State University, University Park, Pennsylvania 16802 SUMMARY we examined the reactive patterns of cancer patient sera with antigens isolated from cells transformed by these vi Antigens isolated from herpes simplex virus type 1, muses. Our studies reveal a positive correlation between herpes simplex virus type 2, on cytomegalovinus-trans sera of patients with diagnosed squamous cell carcinoma formed hamster cells were tested against 66 semafrom non and antigens isolated from both HSV-1- and HSV-2-trans cancer individuals or patients with different types of cancer. farmed cells. By use of the microcomplement fixation procedure to quan tify all antigen-antibody interactions, it was observed that MATERIALS AND METHODS 94% (p < 0.001) of all semafrom patients with squamous cell carcinoma reacted with antigens from herpes simplex virus Cells. Hamster cells transformed by HSV-1 [14-012-8-1; type 1-transformed cells, while 84% (p < 0.001) of the same (9)], HSV-2 [333-8-9 (8)], on cytomegalovirus [CX-90-3B, T2 sena reacted with antigen preparations from herpes simplex (2)] were acquired from F. Rapp, M. S. Hershey Medical virus type 2-transformed cells. When semafrom patients with Center, Hershey, Pa., while cell cultures of normal non adenocancinoma, sarcoma, liposarcoma, and melanoma transformed hamster cells were prepared from 13-day-old were tested against these antigens, there was no significant embryos (Lakeview Hamster Colony, Newfield, N. J.). All difference in their reactivity from sera of noncancem pa cells were propagated and maintained in Dulbecco's modi tients. When sera from all individuals (normal and cancer) fied Eagle's medium; 10% fetal calf serum; 0.075% NaHCO3; were tested against antigens from cytomegalovirus-trans penicillin, 100 units/mI; and streptomycin, 10 pg/mI. formed cells, no significant reaction pattern developed. Tumor Antigen Preparation. Antigens extracted from all These studies are the first to describe the isolation of a cells, bath normal and transformed, were prepared from reactive tumor-associated protein from herpes simplex vi cultures of the different cell types used. Monolayer cultures mus-transfommedcells. of each cell type were rinsed 3 times with cold (4°)Eagle's basal medium containing 0.11% NaHCO3, scraped into the same medium, and recovered by centnifugation. The cell INTRODUCTION pellet was resuspended as a 10% solution in cold 0.85% NaCI solution, and the cells were subjected to repeated A variety of evidence has accumulated which associates sequential sonic disruption (3, 2, 3, 4 mm at 4°,20kc MSE members of the Herpesvirus group with different cancers in disintegrator) followed by ultracentnifugation (100,000 x many species (23). Of particular interest are studies that 4°).Thesupemnatants were combined, and protein concen suggested a relationship between HSV-13 and human oral tration was determined by the method of Lowry et al. (17), cancers (30) on HSV-2 and human uterine cervical carci after which this crude extract was fractionated by discontin noma (19). Although many of these studies are semoepide uous gradient polyacrylamide electrophoresis (12, 29). Sol miological (1, 26), the oncogenic capability of both HSV-1 uble antigens in gel region B (lower 45% of gel) were eluted and HSV-2 has been demonstrated in vitro by transforming and concentrated by Diaflo filtration (UM1Ofilter, 4°)diluted cells with inactivated on temperature-sensitive mutants of 100-fold with cold 0.85% NaCI solution, and reconcentrated the virus (8, 9, 14, 24, 28). Considering the relationship of to 1.25 mg/mI. The final concentrate was used in the MCF HSV-1 and HSV-2 to certain carcinomas of man and the test as described below. The tumor antigen fraction iso availability of cells transformed in vitro by HSV-1 or HSV-2, lated from HSV-1-transfommed cells is referred to as M-1, while antigens from HSV-2-transformed cells are referred to , Supported by Contract NO1-CB-43984 from the National Cancer Institute. as M-2. Antigens from cytomegalovmmus-transformed cells 2 To whom requests for reprints should be addressed, at the Department of Microbiology and Cell Biology, Pennsylvania State University, 5101 Frear are designated Cx90, and extracts of nontransfommed ham Building, University Park, Pa. 16802. stemembryo cells were not labeled and served the dual pun 3 The abbreviations used are: HSV-1 , herpes simplex virus type I ; HSV-2, pose of fetal antigen preparation and tissue controls in all herpes simplex virus type 2; MCF, microcomplement fixation; M-1, tumor antigen from HSV-1-transformed cells; M-2, tumor antigen from HSV-2-trans assays. formed cells; Cx90, cell extract from cytomegalovirus-transformed cells; Virus Antigen Preparation. Vinion antigens were pre HEL, cell extract from human embryonic lung cells; HSV, herpes simplex virus. pared by infecting HEL cells with HSV-1 (Seibert strain) or Received June 11, 1976; accepted August 17, 1976. HSV-2 (316-D strain) at a multiplicity of infection of 5. After 4394 CANCERRESEARCHVOL. 36 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1976 American Association for Cancer Research. Reaction of Herpes-transformed Cell Antigens with Cancer Sera 24 hr, newly synthesized infectious virus and accompanying known negative serum, serum controls, tissue antigen con infected membranes were separated from large cell debris trols, complement controls, and Kalmer (16) saline con by differential centrifugation. The partially purified virus trols. These precautions were sufficient to establish anti plus infected cell membranes were pelleted by centnifuga complementary activity of any reagent used in each test. tion (100,000 x g, 1 hr) and washed repeatedly with Kol Readings of percentage hemolysis of each microtiter well men's (16) saline. The virus pellet and membranes were solu were made using a test reading mirror which held 1 dispos bilized by extensive sonic disruption, the protein cancentra able plate and 1 row of 12 microtiter wells cut from a tion was determined, and the extract was stored at —60°.disposable plate containing 11 color standards from 0 to Uninfected HEL cells processed as described above served 100% hemolysis. A positive MCF reaction is defined as the as noninfected tissue culture control for vimion antigen in highest serum dilution that resulted in greater than 10% the MCF test. fixation of complement oventhe controls. It should be noted Test Sera. A total of 66 coded sera were obtained from that 10% is a minimum value and was selected in order to patients at the M. S. Hershey Medical Center, The Pennsyl provide sera from noncancer patients every opportunity to vania State University, College of Medicine, Hershey, Pa., react with these antigen preparations. However, the results and the Surgery Branch of the National Cancer Institute, to be presented were also analyzed at the 20 to 40% fixation NIH. These sena included samples from patients diagnosed level, and the data did not change significantly. Clinical as squamous cell carcinoma of uterine cervix, vulva, larynx, information for each serum was withheld until all sera were palate, lip, and tonsil; also included were senafrom patients completely and repeatedly analyzed (a minimum of 3 times with adenocarcinoma of breast, lung, cervix, endometnium, with each serum for all antigens). These results were statis and ovary, as well as semafrom individuals without cancer. tically processed by the x2 or Student's t test. When received, all sera were heat inactivated (56°,30mm), diluted with Kolmer's (16) saline, and stored at —20°. RESULTS MCF. M-1, M-2, Cx90, and fetal antigens were used in a modified MCF test (22) at a concentration of 40 pg/mI (1.0 The results of this study begin with reactive patterns of pg/reaction volume), while vinion antigens were used at a antigens M-1, M-2, Cx90, HSV-1, and HSV-2 with semafrom concentration of 20 @g/ml(0.5jig/reaction volume). Stand patients with squamous cell carcinoma of the uterine cervix ardization of all reagents (hemolysin, complement, and an (Tables 1, 5, and 6). When these patients' semawere reacted tigens) was performed on a regular basis as well as with with tumor antigens M-1 on M-2, 20 of 22 [91%, p < 0.001 each new lot of antigen. A disposable “U―platecontaining (Tables 1 and 5)] and 19 of 22 [86%, p < 0.001 (Tables 1 and 96 wells was used to examine simultaneously several dilu 5)], respectively, reacted in a positive fashion. In contrast, tions of each unknown serum, 1 known positive serum, 1 the reactivity of these semawith extracts of Cx90 was only Table1 Reaction pattern of sera from female patients with squamous cell carcinoma of the uterine cervix when exposed to antigens extracted from HSV-1(M-1), HSV-2(M-2), and cytomegalovirus-transformed cells, as wellas HSV-1and HSV-2 virion antigensMCF titers0Sample HSV-2952Age (yr) Race Term M-1 M-2 Cx90 HSV-1 56 C'@ Preoperative 8 16 32 256 256 1094 60 C Postoperative 4 8 0 64 64 226 49 C Postoperative 4 16 0 64 64 316 56 C Postoperative 4 2 0 64 64 855 46 C Preoperative 8 8 0 128 128 1028 46 C Preoperative 4 8 0 32 32 729 58 C Preoperative 32 32 0 1024 512 944 49 N Preoperative 8 8 0 64 64 75623 53 C Preoperative 8 8 0 4 0 73434 71 C Preoperative 4 4 0 64 64 79621 57 C Preoperative 8 0 0 128 32 73251 45 C Preoperative 2 4 0 32 64 73604 59 C Preoperative 4 4 0 64 32 75416 29 C Preoperative 4 4 0 128 128 76234 77 C Preoperative 4 16 0 512 512 80352 58 C Preoperative 4 16 0 64 64 78757 48 C Postoperative 4 4 0 64 64 85091 56 C Preoperative 4 2 0 64 256 82736 58 C Preoperative 0 4 0 64 32 84956 61 C Preoperative 0 0 0 128 256 73765 66 C Preoperative 8 16 0 32 64 84512 53 0 Preoperative 4 0 0 128 64 a Numerical titers express the reciprocal of the highest dilution of serum that fixed greater than 10% of complement when comparedto controls.
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