Doctor of Philosophy
Microbiology & Immunology
B . K. Murray
Many lines of evidence exist associating herpes simplex virus (HSV) with the development of carcinoma. Data from human studies includes seroepidemiologic studies of carcinoma patients and the localization of viral markers in human cancers. Experimental studies include in vitro transformation of cultured cells and viral induced alterations of host DNA. Much of this evidence is anecdotal or associative in nature and does not prove a cause and effect. The purpose of this research was to investigate the oncogenic potential of HSV type 2 (HSV-2) in vivo and in vitro.
An in vivo mouse model for lib carcinogenesis was designed to combine HSV-2 infection, ultraviolet (UV) exposure and tetradecanyl-phorbolacetate (TPA) application. Preliminary studies showed that HSV-2 inoculation onto abraded mouse lips was capable of causing vesicular ulcerative lesions. These lesions healed completely after 10-14 days. Repeated herpetic lip infections failed to induce tumors. UV-irradiation delivered to the lesion site daily for 6 minutes at 42 ergs/mm2/s on days 3 through 6 postinfection caused hyperkeratosis, acanthosis and dysplasia to develop in several lips; while the same UV exposure delivered by itself failed to alter the histologic appearance. The addition of related TPA application to the HSV + UV regimen promoted tumor emergence. Thirty-two of 156 BALB/c mice developed tumors. Although the majority were papillomas, six were squamous cell carcinomas. These tumor bearing mice had increased HSV specific antibody titers. HSV antigens were shown to be present in outgrowths from explanted tumors as well as in tumor biopsies by immunoperoxidase staining with HSV specific antiseras.
Another series of in vivo studies showed that HSV lip infection initiated prior to or during repeated chemical carcinogen (dimethylbenzanthracene/DMBA) application was capable of reducing the incidence of tumors as compared to DMBA application without superimposed HSV infection. Comparison of results from this system and those from the HSV + UV + TPA system emphasized the fact that the lytic capability of HSV must be inhibited for the virus to express its oncogenic potential. It was hypothesized that the in vivo UV-irradiated HSV acted as the inducer and TPA as the promoter, analogous to the classical two-state carcinogenesis model. Since neither the HSV infection by itself, the UV exposure by itself, the TPA application by itself nor any combination of two induced the development of squamous cell carcinomas, HSV was considered a carcinogen with UV-radiation.
An analogous tripartite system was devised in vitro. Hamster embryo cells were infected with HSV-2 irradiated for 6, 7, or 8 minutes at 42 ergs/mm2/s. Twenty-one days postinoculation transformed foci had developed with frequencies (FFU/PFU) of 2.9 x 10 -7, and 3.9 x 10 -7, respectively. This represents 0.9, 3.0, and 1.3 FFU/10 6 cells. The 7 minute transformation frequencies were further increased to 1.7 x 10 -6 by the addition of TPA to the growth media (0.1 ug/ml) 48 hours postinoculation. This represents 5.1 FFU/10 6 cells. Thus, TPA was capable of tumor promotion in vivo and increasing transformation efficiency in vitro. Furthermore, TPA was shown by two dimensional gel electrophoresis to increase protein synthesis in transformed cells and by cell sorter analysis to increase cellular replicative activity.
A transformed cell line, 3-26-7#1, derived from a UV-inactivated HSV transformation experiment, was shown to possess HSV antigens by immunoperoxidase staining with specific antisera and to be oncogenic when injected into newborn hamsters. Sera from the tumor bearing animals had HSV neutralizing antibodies and when used as the primary antisera in the immunoperoxidase stain could detect antigens in HSV infected cells and outgrowths from explanted hamster tumors. Another transformed cell line, 333-8-9, was shown by in situ hybridization to have HSV specific mRNA in its cytoplasm.
These data reveal UV-irradiated HSV capable of tumor induction in vivo and of transforming hamster cell in vitro. In these systems, HSV can be considered a cocarcinogen.
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