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[CANCER RESEARCH 33, 1527-1534, June 1973] Transformation of Hamster Embryo Fibroblasts by Herpes Simplex Viruses Type 1 and Type 21 Fred Rapp2 and Ronald Duff Department of Microbiology. College of Medicine, The Milton S. Hershev Medical Center, The Pennsylvania Slate University, Hershev, Pennsylvania 17033 Summary Hamster embryo fibroblast cells can be transformed after infection by ultraviolet light-irradiated herpes simplex viruses type 1 (HSV-1) or type 2 (HSV-2). Two ofl2 strains of HSV-1 and 7 of 15 strains of HSV-2 induced cell transformation. Transformed cells from two strains of HSV-2 and one strain of HSV-1 have been tested for oncogenicity in newborn Syrian hamsters. One cell line transformed by HSV-2 strain 333 was found to be oncogenic. The remaining two cell lines were not oncogenic. Herpesvirus antigens were found in the cytoplasm and on the surface of the transformed cells. Neutralizing antibodies were produced in hamsters developing tumors after the injection of the HSV-2-transformed cells. Weanling ham sters that were preimmunized by injection of HSV-1 or HSV-2 did not develop transplantation immunity to the HSV-2-transformed cells as a result of this preimmunization. HSV-2-transformed cells metastasized after injection into hamsters. The number of métastaseswas not inhibited by HSV-1 or HSV-2 immunization but was actually enhanced. Immunization of hamsters with simian virus 40 inhibited métastasesbut did not reduce the rate of primary tumor development. Introduction Four of the major groups of viruses containing DNA induce in vitro transformation and in vivo induction of tumors (2, 9). The most likely candidates for viruses oncogenic in humans appear to be members of the herpesvirus group. Several lines of evidence have been documented that link these viruses to cancer in both lower animals and man. The best documented case of the expression of oncogenic potential by a herpesvirus is found in a disease of chickens, Marek's disease (4, 22). The infection of a young chicken with a herpesvirus induces a highly proliferative lymphosarcoma that ultimately results in the death of the animal. In addition to the direct demonstration by virus injection that a herpesvirus is responsible for this disease, further evidence that Marek's disease is caused by a ' Presented at the American Cancer Society Conference on Herpesvirus and Cervical Cancer, December 8 to 10, 1972, Key Biscayne, Fla. This work was conducted under Contract 70-2024 within the Special VirusCancer Program of the National Cancer Institute, NIH, USPHS. 2Presented by. JUNE 1973 herpesvirus comes from the observation that the disease can be prevented by the injection of a similar attenuated virus that does not induce the neoplasia (23). More recently a herpesvirus has been implicated in lymphomas that can be induced in monkeys (16 19, 29). This virus, Herpesvirus saimirÃ-, can be isolated from squirrel monkeys (SaimirÃ-sciureus) without a tumor; how ever, it induces lymphomas or lymphocytic leukemia in marmosets (Saguinus oedipus, Saguinus fusciollis, and Saguinus nigricollis), owl monkeys (Aotus albifrons), and cinnamon ringtail monkeys (Cebus albifrons). In addition to these demonstrations of herpesvirus oncogenicity in ani mals, the Epstein-Barr virus of humans has been associated with Burkitt's lymphoma. Cells derived from Burkitt lym phomas contain virus antigens that can be detected by immunofluorescence and herpes-like particles that can be observed with the electron microscope (10, 13). The virus that can be isolated is not highly infectious but has been shown to induce the in vitro transformation of leukocytes (15). A very similar, and probably identical, virus has been shown to be associated with infectious mononucleosis and is now thought to be the causative agent ofthat disease (14). A 2nd human herpesvirus which has been associated with human neoplasia is HSV-2.3 The original observation that implicated this virus in cancer was the result of seroepidemiological studies. It was demonstrated that women with cervical carcinoma have circulating antibodies directed against the HSV-2 more often than women without the disease (21, 24). HSV-2 has also been isolated from cervical carcinoma cells and HSV-2 antigens can be demonstrated in similar cells by immunofluorescent techniques (1, 27). However, HSV's when injected into experimental animals rarely, if ever, induce tumors (20, 25). Recently, in our laboratory we have demonstrated that HEF cells can be transformed by UV-irradiated HSV-2 and that these cells are oncogenic when injected into newborn hamsters (8, 9). Transformation of Hamster Embryo Cells HSV-1 and HSV-2 are extremely cytopathic in tissue culture cells that have been infected by the virus. Any potential oncogenic transformation would therefore be masked by the cell death that results from HSV infection. 3The abbreviations used are: HSV-2. herpes simplex virus type 2; HSV. herpes simplex virus; HEF, hamster embryo fibroblast; HSV-1, herpes simplex virus type 1; SV40, simian virus 40. 1527 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1973 American Association for Cancer Research. Fred Rapp and Ronald Duff The elimination of cell death induced by HSV-1 and HSV-2 was accomplished by UV irradiation'(42 ergs/sec/sq cm) of the virus before infection of the host cell. Inactivated HSV-1 and HSV-2 were absorbed onto HEF cells in vitro while the cells and virus were in suspension. Three to 4 weeks after the HSV infection, transformed cells appeared in the culture monolayer. These cells could then be isolated from the monolayer and grown in culture into cell lines. Under the conditions used, both HSV-1 and HSV-2 have transformed HEF in vitro (Table 1). Seven of 15 HSV-2 strains induced HEF cellular transformation and 2 of 12 HSV-1 strains also transformed HEF cells. The morphology of the transformed hamster embryo cells has been constant depending upon the type of HSV used to transform the cells. Cells transformed by HSV-2 were usually of mixed morphology (Fig. 1). Three types of cellular morphology were generally seen: (a) a fibroblastic cell, (b) a giant cell, and (c) a rather primitive mesenchymal-like cell. In contrast to this type of transfor mation induced by HSV-2, HSV-1-transformed cells usu ally exhibited an epithelial-like morphology (Fig. 2). In addition to the epithelial cells, many giant cells were also observed in HSV-1-transformed HEF cells. Antigenic Characteristics of HSV-transformed Table 2 HSV-specific antigens on the surface of HSV-2-iransformed hamster embrvo cells SerumAmi of cells reacting type333-8-9HEF333-8-9HEF333-8-9 at surface60.912.260.39.37.41 HSV-1Ami HSV-2Weanling hamsterCell HEF% Table 3 Induction of tumors in newborn hamsters by the injection of hamster cells after in vitro transformation by U V-irradialed HS V-2 strain 333 hamsterswith of (wk)10 period Cell line333-8-9333-2-20333-2-21333-2-26333-2-29% tumors36100KM)10098Latent 162 42-42-42-7 Cells Cells that have been transformed by either DNA or RNA oncogenic viruses usually contain virus-specific antigens. For example, the transformation of a cell by the SV40 usually results in the induction of the SV40 T or tumor antigen in the nucleus and in the induction of the SV40 S antigen on the surface of the cell (3, 24, 28). These antigens are usually demonstrated by the use of immunofluorescence techniques. Cells that have been transformed after infection by HSV-1 or HSV-2 were tested by indirect immunofluores cence techniques for the presence of HSV-specific antigens. When anti-HSV serum that had been prepared in weanling hamsters was absorbed to acetone-fixed HSV-transformed cells, a cytoplasmic fluorescence was detected within these cells (Fig. 3). The diffuse fluorescence observed throughout the cytoplasm was seen in 5 to 25% of the transformed HEF cells. antigen reacted with both antiHSV-1The and cytoplasmic anti-HSV-2 sera.^ serum stained only a small percentage of the transformed cells exposed to this reagent. Characteristics of HSV-transformed Cells in Vivo The transformed cells (2 x IO5 cells/hamster) were injected into weanling and newborn Syrian hamsters. No tumors were induced in weanling hamsters by any of the 7 cell lines tested. All hamster cell lines transformed by the HSV-2 strain 333 were oncogenic after injection into newborn hamsters. However, cell lines that had been established following transformation by this strain of HSV-2 differed with respect to their relative oncogenic potential. As shown in Table 3, 1 cell line (333-8-9) was of relatively low oncogenic potential after injection into new born hamsters. However, the remaining 4 cell lines that were transformed by strain 333 (333-2-20, 333-2-21, 333-2-26, and 333-2-29) induced tumors with greater fre The HSV-2 transformed cells were also examined for the quency and with shorter latent periods than did the 333-8-9 presence of herpesvirus-specific antigens on the surface by cells. In contrast to the lines transformed by strain 333, cells that were transformed by HSV-2 strain 332 and HSV-1 utilizing indirect immunofulorescence techniques and un fixed cells. As shown in Table 2, significant numbers of strain KOS were not oncogenic when injected into newborn these transformed cells demonstrated fluorescence on their hamsters. From these results we have now classified HSVsurface after treatment with anti-HSV serum. Normal cells transformed cells into 3 types with regard to their oncogenic reacted in very low numbers and normal weanling hamster potential: high, low, and nononcogenic. Sera from tumor-bearing hamsters were tested for anti bodies against HSV's. It was found that a significant Table I number of the tumor-bearing hamsters developed neutraliz Transforma/ion of HEF by isolates of HSV-1 and HSV-2 ing antibodies directed against HSV-2. Sera that neutral isolateswith of ized HSV-2 did not neutralize HSV-1 as efficiently in most ofisolatestested1215No.detectabletransformationpotential27 cases (Chart 1). It was also found that the development of neutralizing antibodies was inversely proportional to the Virus typeHSV-1HSV-2No. latent period of tumor development in the host hamster. The cell lines transformed by HSV-2 strain 333 that had a shorter latent period in the newborn hamsters did not induce 1528 CANCER RESEARCH VOL. 33 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1973 American Association for Cancer Research. Cellular Transformation by HSV Transplantation Immunity Studies with HSV-2-transformed Cells 100. to OC O en D in o oc O HSV 333-8-9 (LSH-1) TYPE SERUM 21 21 A 21 B 21 C 21 D E Chart 1. Neutralization of HSV-2 and HSV-1 by sera obtained from hamsters with tumors induced by an s.c. injection of HSV-2-transformed cells (333-8-9, LSH-I). All hamsters were given injections of the trans formed cells at 3 to 4 weeks of age. The results are expressed as the percentage of virus survivors after incubation of 1000 plaque-forming units of HSV with 1 ml of a 1/10 serum dilution. neutralizing antibodies with as great a frequency as did the cell line with the longer latent period (Table 4). The location of the antigens that induced the neutralizing antibody response in the tumor-bearing hamsters is not clear at the present time. The surface or cytoplasmic antigens which have been observed may be the antigens that induce this immune response. However, in a few of the tumor cells immature HSV-like particles have been observed and this low-level production of presumably noninfectious HSV may result in the induction of HSV-2-neutralizing antibodies (ID- Neutralization of HSV-1 The study of the characteristics of transplantation im munity was not possible with the primary transformed cell lines because they failed to induce tumors in weanling animals. For determination of whether weanling hamsters could be used, transformed cells that had been passed 1time in a newborn hamster were tested for their oncogenic potential in weanling hamsters (Chart 2). After 1 passage in a newborn hamster the 333-8-9 cell line was found to have increased oncogenicity to the point where it induced tumors when small numbers of cells were injected into weanling hamsters. A 2nd transformed cell line (333-2-26) was also tested for its oncogenic potential in weanling hamsters after the cell line had been passed once in a newborn hamster. As previously shown (Table 3), this cell line was much more oncogenic when injected into newborn hamsters than was the 333-8-9 cell line. The 333-2-26 cell line also showed increased oncogenic potential after 1 animal passage and induced tumors in weanling hamsters (Chart 2). However, it was not as oncogenic as was the 333-8-9 cell line after passage in a newborn hamster. It can be postulated from this result that the longer in vivo latent period of the 333-8-9 cell line resulted in the selection of cells with a higher oncogenic potential than did the shorter latent period of the 333-2-26 cells. Both cell lines induced metastatic tumors in the lungs of animals after s.c. injection of the cells (Fig. 4). Métastaseswere also found in the liver, gastrointestinal tract, and kidneys of the injection-treated animals. The development of métastasesby tumor-bearing hamsters indicated that, at most, a very low level of tumor immunity was induced by the cells in the host hamster. As a test of this hypothesis, weanling hamsters were immunized by at tenuated HSV-1 strain 35 (25). The injection of this strain of HSV-1 did not result in the death of the hamster. In addition to the injection of HSV-1 strain 35, hamsters were also immunized by the DNA tumor virus SV40, UVirradiated 333-8-9 cells, and UV-irradiated HEF cells. Each animal received 3 injections of an immunizing agent at weekly intervals followed 1 week later by the injection of the tumor cells. As sfÃ-ownin Chart 3 the preimmunization of hamsters by HSV-1 strain 35, SV40, 333-8-9 cells, or HEF cells did not inhibit the development of primary tumors. Table 4 and HSV-2 by sera from hamsters HSV-2-transformed cells bearing tumors induced by Anti-HSV-1 sera"/total Anti-HSV-2 sera"/total positive6064.312.541.213.8Positive positive10035.762.570.675.9 line333-8-9333-2-20333-2-21333-2-26333-2-29Positive Cell sera tested3/59/142/167/174/29% tested5/55/1410/1612/1722/29% sera " Positive sera are defined as the sera that neutralized of HSV-2 at a I/10 serum dilution. JUNE 1973 at least 50% of 1000 plaque-forming units 1529 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1973 American Association for Cancer Research. Fred Rapp and Ronald Duff métastasesby the HSV-transformed cells when animals were immunized with 3 injections of this virus. Since it has been previously shown that SV40-transformed cells contain new surface embryonic antigens (5, 7), it can be postulated that the inhibition of métastasesmight be a result of the stimulation of an immune response against hamster embry onic antigens. The enhancement of métastasesafter the injection of HSV-1 is possibly due to the presence of blocking antibodies that have been previously described in other tumor systems (12). These results that demonstrate the enhancement and inhibition of métastasesafter an s.c. injection of 333-8-9 cells have been repeated utilizing a 2nd HSV-2-transformed cell line, the 333-2-26 line (Table 5). Although the frequency of métastaseswas somewhat less with the 333-2-26 line, the pattern of métastaseswith re gard to the inhibition by SV40 and the enhancement by HSV-1 was essentially the same. o 0- Discussion 10 WEEKS AFTER 11 12 CELL CHALLENGE Charl 2. The rate of tumor induction in weanling Syrian hamsters after the injection of HSV-2 transformed hamster cells. Hamsters received s.c. injections of 333-8-9 cells (•)or 333-2-26 (O). TPD/50, 50% tumorproducing dose. Since it was possible that the attenuation of HSV-1 strain 35 destroyed its potential for the induction of tumor immunity, nonattenuated strains of HSV-1 and HSV-2 were also injected after UV irradiation to destroy virulence. However, the results using UV-inactivated HSV-1 and HSV-2 were the same as the results obtained using the HSV-1 strain 35. No detectable inhibition of the primary tumors was ob served using UV-irradiated HSV-1 or HSV-2. If extremely low levels of transplantation immunity were induced by injections of HSV-1 or HSV-2 in weanling hamsters, this low level of immunity might be detected by inhibition of métastasesrather than by inhibition of the primary tumor. To answer this question, animals were immunized by HSV-1 strain 35, SV40, UV-irradiated 333-8-9 cells, and UV-irradiated HEF cells. The preimmunization of weanling hamsters by HSV-1 resulted in an enhancement of métastasesin these animals (Table 5). The immunization of weanling hamsters by UV-irradiated 333-8-9 cells and HEF cells resulted in a significant decrease in the number of métastases.SV40 completely inhibited 1530 The results summarized in this communication provide evidence for the association of HSV-2 as well as HSV-1 with the transformation of normal hamster cells into morphologically altered cells. In some cases this morpho logical transformation was accompanied by the conversion of the normal cells into cells with oncogenic potential. However, in all cases HSV antigens could be demonstrated in the cytoplasm and on the surface of the cells. Recently, the transformation of human embryonic lung cells has been reported following abortive infection by HSV-2 (6). Neither HSV-1 nor HSV-2 has been conclusively shown to trans form human cells in our laboratory. However, experiments now in progress are designed to demonstrate this. The frequency of oncogenic transformation by HSV-1 or HSV-2 still remains to be completely delineated. Cell lines that have been transformed by 2 strains of HSV-2 and 1 strain of HSV-1 have been tested for oncogenic potential. Only 1 group of 5 transformed cell lines that was trans formed by the HSV-2 strain 333 was oncogenic after injection into newborn hamsters. After further extensive testing of many cell lines transformed by several virus strains it can be determined whether this oncogenic conver sion is a relatively rare characteristic of HSV-1 and HSV-2 strains or whether it occurs at a relatively high frequency. The final observation described in this communication is that HSV-2-transformed cells do not contain levels of virus-specific transplantation antigens that are found in most cell lines transformed by other DNA viruses. This apparent lack coupled with failure to stimulate the immune response of the host animal is probably responsible for the high rate of métastasesthat is found in HSV-2 tumor-bear ing animals. The procedure described to control enhance ment and repression of these métastasessuggest that the HSV-2-transformed cells may represent a model system for the experimental control and characterization of métastases in mammals. CANCER RESEARCH VOL. 33 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1973 American Association for Cancer Research. Cellular Transformation by HSV 10*1 e io 10 WIIK1 «Ft« CHI CHAUINOE WIIKS »ft« CUI CHAUINOI Chart 3. The rate of tumor induction by HSV-2-transformed cells in weanling Syrian hamsters after immunization. Immunizing agents in Chart ÃŒA were HSV-1 (O). SV40 (•),or Medium 199control (x). Immunizing agents in Chart 3B were UV-irradiated HEF cells (•).UV-irradiated 333-8-9 cells (D), and Medium 199 control (x). Hamsters in each immunization group received 3 immunizing injections at 1-week intervals. The animals were challenged with 333-8-9 cells I week after the final immunizing injection. TPD/50. 50% tumor-producing dose. Table 5 Effect of immunization on the development of métastases¡nweanling hamsters of with lung hamsters with given injections27403840343540404039Hamsters Cell line333-8-9333-2-26Immunizing agentHSV-1"SV40"333-8-9'HEF"Medium' métastases13032760124% métastases48.107.95.020.617.102.55.010.3 con trolHSV-1-SV40*333-8-9'HEF"Medium' con trolTotalhamsters " Hamsters in this group were immunized by 3 injections of IO6plaque-forming units of HSV-1 strain 35. " Hamsters in this group were immunized by 3 injections of IO6plaque-forming units of SV40. ' Hamsters in this group were immunized by 3 injections of 10" 333-8-9 cells that were UVirradiated for 3 min (42 ergs/sec/sq cm). " Hamsters in this group were immunized by 3 injections of 10e HEF cells that were UV-ir radiated for 3 min (42 ergs/sec/sq cm). ' Hamsters in this group were given 3 injections of Medium 199as a control. JUNE 1973 1531 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1973 American Association for Cancer Research. Fred Rapp and Ronald Duff Acknowledgments The authors wish to thank Myron Katz and Doreen Lakatosh for their valuable technical assistance. References 1. Aurelian, L., Royston, I., and Davis, H. J. Antibody to Genital Herpes Simplex Virus: Association with Cervical Atypia and Carcinoma in Situ. J. Nati. Cancer Inst., 45: 455 464, 1970. 2. Black, P. H. Recent Advances in the Study of Oncogenic Viruses. New Engl. J. Med., 275: 377 383, 1966. 3. Black, P. H., Rowe, W. P., Turner. H. C, and Huebner, R. J. A Specific Complement-Fixing Antigen Present in SV40 Tumor and Transformed Cells. Proc. Nati. Acad. Sci. U. S.. 50: 1148 1156, 1963. 4. Churchill, A. E., and Biggs, P. M. Agent of Marek's Disease in Tissue Culture. Nature, 215: 528 530. 1967. 5. Coggin, J. H., Ambrose, K. R., and Anderson, N. G. Fetal Antigen Capable of Inducing Transplantation Immunity against SV40 Ham ster Tumor Cells. J. Immunol., 105: 524 526, 1970. 6. Darai, G., and Munk, K. Human Embryonic Lung Cells Abortively Infected with Herpesvirus hominis Type 2 Show Some Properties of Cell Transformation. Nature, 241: 268 269, 1973. 7. Duff. R., and Rapp. F. Reaction of Serum from Pregnant Hamsters with Surface of Cells Transformed by SV40. J. Immunol.. 105: 521 523, 1970. 8. Duff, R., and Rapp, F. Oncogenic Transformation of Hamster Cells after Exposure to Herpes Simplex Virus Type 2. Nature. 233: 48 50, 1971. 9. Duff, R., and Rapp, F. Properties of Hamster Embryo Fibroblasts Transformed in Vitro after Exposure to Ultraviolet Irradiated Herpes Simplex Virus Type 2. J. Virol., 8: 469 477. 1971. 10. Epstein, M. A., Achong, B. G.. and Barr. Y. M. Virus Particles in Cultured Lymphoblasts from Burkitt's Lymphoma. Lancet. /. 702 703, 1964. 11. Glaser, R., Duff, R. G., and Rapp, F. Ultrastructural Characteri/ation of Hamster Cells Transformed following Exposure to Ultravioletirradiated Herpes Simplex Virus Type 2. Cancer Res., 32: 2803 2806. 1972. 12. Hellström,L, and Hellström,K. E. Studies on Cellular Immunity and Its Serum-Mediated Inhibition in Moloney-Virus-Induced Mouse Sarcomas. Intern. J. Cancer, 4: 587 600, 1969. 13. Henle, G., and Henle. W. Immunofluorescence in Cells Derived from Burkitfs Lymphoma. J. Bacteriol., 91: 1248 1256, 1966. 14. Henle, G., Henle, W., and Diehl, V. Relation of Burkitt's TumorAssociated Herpes-Type Virus to Infectious Mononucleosis. Proc. Nati. Acad. Sei. U. S., 59: 94 101, 1968. 15. Henle, W., Diehl, V.. Kohn, G., Zur Hausen. H., and Henle. G. Herpes-Type Virus and Chromosome Marker in Normal Leukocytes after Growth with Irradiated Burkitt Cells. Science, 157: 1064 1065, 1967. 1532 16. Hunt. R. D.. Meléndez, L. V., King, N. W.. Gilmore, C. E., Daniel. M. D., Williamson, M. E., and Jones, T. C. Morphology of a Disease with Features of Malignant Lymphoma in Marmosets and Owl Monkeys Inoculated with Herpesvirus saimirÃ-.J. Nati. Cancer Inst.. 44:447 465, 1970. 17. Meléndez, L. V., Daniel, M. D., Hunt, R. D., Fraser, C. E. O., Garcia, F. G., King. N. W., and Williamson. W. E. Herpesvirus saimirÃ-.V. Further Evidence to Consider This Virus as the Etiological Agent of a Lethal Disease in Primates Which Resemble a Malignant Lymphoma. J. Nati. Cancer Inst., 44: 1175 1181, 1970. 18. Meléndez.L. V., Daniel. M. D., Hunt, R. D., and Garcia, F. G. An Apparently New Herpesvirus from Primary Kidney Cultures of the Squirrel Monkey (SaimirÃ-sciureus). Lab. Animal Care, 18: 374 381. 1968. 19. Meléndez. L. V., Hunt. R. D., Daniel, M. D., Fraser, C. E. O., Garcia, F. G., and Williamson, M. E. Lethal Reticuloproliferative Disease Induced in Cebus alhifronx Monkeys by Herpesvirus saimirÃ-.Intern. J. Cancer, 6:431 435, 1970. 20. Nahmias, A. J., Naib, Z. M.. Josey, W. E.. Murphy, F. A., and Luce. C. F. Sarcomas after Inoculation of Newborn Hamsters with Herpesvirus hominis Type 2 Strains. Proc. Soc. Exptl. Biol. Med., 134: 1065 1069, 1970. 21. Naib, Z. M., Nahmias, A. J., Josey, W. E., and Kramer, J. H. Genital Herpetic Infection: Association with Cervical Dysplasia and Car cinoma. Cancer. 23: 940 945. 1969. 22. Nazerian, K., Solomon, J. J.. Witter, R. L., and Burmester, B. R. Studies on the Etiology of Marek's Disease. II. Finding of a Herpesvirus in Cell Culture. Proc. Soc. Exptl. Biol. Med., 127: 177 182, 1968. 23. Okazaki, W., Purchase. H. G.. and Burmester, B. R. Protection against Marek's Disease by Vaccination with a Herpesvirus of Turkeys. Avian Diseases, /4:4I3 429, 1970. 24. Rapp, F., Butel, J. S., and Melnick, J. L. Virus-induced Intranuclear Antigen in Cells Transformed by Papovavirus SV40. Proc. Soc. Exptl. Biol. Med., 116: 1131 1135, 1964. 25. Rapp. F.. and Falk, L. A. Study of Virulence and Tumorigenicity of Variants of Herpes Simplex Virus. Proc. Soc. Exptl. Biol. Med., 116: 361 365, 1964. 26. Rawls, W. E., Tompkins, W. A. F., and Melnick. J. L. The Association of Herpesvirus Type 2 and Carcinoma of the Uterine Cervix. Am. J. Epidemiol., 89: 547 554, 1969. 27. Royston. I., and Aurelian, L. Immunofluorescent Detection of Her pesvirus Antigens in Exfoliated Cells from Human Cervical Car cinoma. Proc. Nati. Acad. Sei. U. S.. 67: 204 212. 1970. 28. Tevethia, S. S., Katz, M., and Rapp, F. New Surface Antigen in Cells Transformed by Simian Papovavirus SV40. Proc. Soc. Exptl. Biol. Med.. 119: 896 901, 1965. 29. Wolfe, L. G., Falk, L. A., and Deinhardt, F. Oncogenicity of Herpesvirus saimirÃ-in Marmoset Monkeys. J. Nail. Cancer Inst., 47: 1145 1162, 1971. CANCER RESEARCH VOL. 33 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1973 American Association for Cancer Research. Cellular Transformation by HSV Fig. I. Photomicrograph of hamster embryo cells transformed by HSV-2 (333-8-9). H & E, x 300. Fig. 2. Photomicrograph of hamster embryo cells transformed by HSV-1 (KOS-6-9). H & E. x 300. JUNE 1973 1533 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1973 American Association for Cancer Research. Fred Rapp and Ronald Duff riv v r^v &7'i t-¿ -A 2¿J /tó-i/MT^ Fig. 3. Photomicrograph of HSV-2-specific cytoplasmic fluorescence in HSV-2-transformed cells (333-8-9 cells). The indirect immunofluorescence technique was used with anti-HSV-2 serum prepared by the injection of weanling Syrian hamsters, x 300. Fig. 4. Photomicrograph of a tumor cell metastasis in the lung of an adult hamster. The animal had received injections s.c. as a weanling with HSV-2-transformed hamster cells, x 125. 1534 CANCER RESEARCH VOL. 33 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1973 American Association for Cancer Research. Transformation of Hamster Embryo Fibroblasts by Herpes Simplex Viruses Type 1 and Type 2 Fred Rapp and Ronald Duff Cancer Res 1973;33:1527-1534. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/33/6/1527 Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at [email protected]. To request permission to re-use all or part of this article, contact the AACR Publications Department at [email protected]. Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1973 American Association for Cancer Research.